Method of treating dopaminergic and GABA-nergic disorders

ABSTRACT

It is shown here that hedgehog proteins possess novel activities beyond phenotype specification. Using cultures derived from the embryonic day 14.5 (E14.5) rat ventral mesencephalon, we show that hedgehog is also trophic for dopaminergic neurons. Interestingly, hedgehog not only promotes dopaminergic neuron survival, but also promotes the survival of midbrain GABA-immunoreactive (GABA-ir) neurons.

This application is a continuation-in-part of U.S. patent applicationSer. No. 08/900,220, filed Jul. 24, 1997, hereby incorporated byreference in its entirety.

BACKGROUND OF THE INVENTION

The individual symptoms of Parkinson's disease have been described byphysicians from the time of Galen, but their occurence as a syndrome wasnot recognized until 1817. In that year James Parkinson, a Londonphysician, published an essay in which he argued that several differentmotor symptoms could be considered together as a group forming adistinctive condition. His observations are interesting not only becausehis conclusion was correct but also because he made his observations inpart at a distance by watching the movements of Parkinsonian victims inthe street of London. Parkinson's disease has been called at differenttimes the shaking palsy or its Latin equivalent, paralysis agitans, butreceived its commoner designation from Jean Charcot, who suggested thatthe disease be renamed to honor James Parkinson's recognition of itsessential nature.

Parkinson's disease is fairly common, estimates of its incidence varyingfrom 0.1 to 1.0% of the population. It is also of considerable interestfor a number of other reasons. First, the disease seems related to thedegeneration of the substantia nigra, and to the loss of theneurotransmitter substance dopamine, which is produced by cells of thisnucleus. The disease, therefore, provides an important insight into therole of this brainstem nucleus and its neurotransmitter in the controlof movement. Second, because a variety of pharmacological treatments forParkinson's disease relive different features of its symptoms to someextent the disease provides a model for understanding pharmacologicaltreatments of motor disorders in their more general aspects. Third,altough Parkinson's disease is described as a disease entity, thesymptoms vary enormously among people, thus making manifest thecomplexity with which the components of movement are organized toproduce fluid motion. Fourth, because many of the symptoms ofParkinson's disease strikingly resemble changes in motor activity thatoccur as a consequense of aging, the disease provides indirect insightinto the more general problems of neural changes in aging.

There are three major types of Parkinson's disease: idiopathic,postencephalitic, and drug-induced. Parkinson's diseases may also resultfrom arteriosclerosis, may follow poisoning by carbon monoxide ormanganese intoxication, or may result from syphillis or the developmentof tumors. As is suggested by its name, the idiopathic cause ofParkinson's disease is not known. Its origin may be familiar, or it maybe part of the aging process, but it is also widely thought that itmight have a viral origin. It most often occurs in people who are over50 years of age. The postencephalitic form originated in the sleepingsickness that appeared in the winter of 1916–1917 and vanished by 1927.Although the array of symptoms wsa bewilderingly varied, such thathardly any two patients seemed alike, Constantin von Economodemonstrated a unique pattern of brain damage associated with a virusinfection in the brains of patients who had died from the disease. Athird of those affected died in the acute stages of sleeping sickness instates either of coma or of sleeplessness. Although many people seemedto completely recover from the sickness, most subsequently developedneurological or psychiatric disorders and parkinsonism. The latencybetween the initial and subsequent occurences of the disease has neverbeen adequately explained. Specify searches for vital particles or virusspecific products in Parkinson patients have revealed no evidence ofviral cause. The third major cause of Parkinson's disease is morerecent, and is associated with ingestion of various drugs, particularlymajor tranquilizers that include reserpine and several phenothiazine andbutyrophenone derivatives. The symptoms are usually reversible, but theyare difficult to distinguish from those of the genuine disorder.

Recently it has been found that external agents can cause symptoms quiterapidly. Langston and coworkers have reported that a contaminant ofsynthetic heroin, MPTP, when taken by drug users is converted into MPPwhich is extremely toxic to dopamine-producing cells. A number of youngdrug users were found to display a complete parkinsonian syndrome afterusing contaminated drugs. This finding has suggested that othersubstances might cause similar effects. Demographic studies of patientadmission in the cities of Vancouver and Helsinki show an increase inthe incidence of patients getting the disease at ages younger than 40.This has raised the suggestion that water and air might containenvironmental toxins that work in a fashion similar to MPTP.

Although Parkinsonian patients can be separated into clinical groups onthe basis of cause of the disease, it is nevertheless likely that themechanisms producing the symptoms have a common origin. Either thesubstrantia nigra is damaged, as occurs in idiopathic andpostencephalitic cases or the activity of its cells is blocked or cellsare killed, as occurs in drug induced parkinsonism. The cells of thesubstantia nigra contain a dark pigment in Parkinson's disease this areais depigmented by degeneration of the melatonin containing neurons ofthe area. The cells of the substantia nigra are the point of origin offibers that go to the basal ganglial frontal cortex and to the spinalcord. The neurotransmitter at the synapses of these projection isdopamine. It has been demonstrated by bioassay of the brains of deceasedparkinsonian patients, and by analysis of the major metabolite ofdopamine, homovanillic acid, which is excreted in the urine, that theamount of brain dopamine is reduced by over 90% and is often reduced toundetectable amounts. Thus the cause of Parkinson's disease has beenidentified with some certainty as a lack of dopamine or in drug inducedcases with a lack of dopamine action.

Certain attempts have been made to treat Parkinson's disease. Oneproposed treatment for Parkinson's disease is Sinemet CR, which is asustained-release tablet containing a mixture of carbidopa and levodopa,available from The DuPont Merck Pharmaceutical Co. Another proposedtreatment for Parkinson's disease is Eldepryl, which is a tabletcontaining selefiline hydrochloride, available from SomersetPharmaceuticals, Inc. Another proposed treatment for Parkinson's diseaseis Parlodel, which is a tablet containing bromocriptine mesylate,available from Sandoz Pharmaceuticals Corporation.

SUMMARY OF THE INVENTION

One aspect of the present application relates to a method for promotingthe survival of dopaminergic or GABAergic neurons by contacting thecells, in vitro or in vivo, with a hedgehog therapeutic or ptctherapeutic in an amount effective increasing the rate of survival ofthe neurons relative to the absence of administeration of the hedgehogtherapeutic or ptc therapeutic.

One aspect of the present application relates to a method for promotingthe survival of neurons of the substantia nigra by contacting the cells,in vitro or in vivo, with a hedgehog therapeutic or ptc therapeutic inan amount effective increasing the rate of survival of the neuronsrelative to the absence of administeration of the hedgehog therapeuticor ptc therapeutic.

In other embodiments, the subject method can be used for protectingdopaminergic and/or GABAergic neurons of a mammal fromneurodegeneration; for preventing or treating neurodegenerativedisorder; for treatment of Parkinson's; for treatment of Huntington's;and/or for treatment of ALS. In embodiments wherein the patient istreated with a ptc therapeutic, such therapeutics are preferably smallorganic molecules which mimic hedgehog effects on patched-mediatedsignals.

Wherein the subject method is carried out using a hedgehog therapeutic,the hedgehog therapeutic preferably a polypeptide including a hedgehogportion comprising at least a bioactive extracellular portion of ahedgehog protein, e.g., the hedgehog portion includes at least 50, 100or 150 amino acid residues of an N-terminal half of a hedgehog protein.In preferred embodiments, the hedgehog portion includes at least aportion of the hedgehog protein corresponding to a 19 kd fragment of theextracellular domain of a hedgehog protein.

In preferred embodiments, the hedgehog portion has an amino acidsequence at least 60, 75, 85, or 95 percent identical with a hedgehogprotein of any of SEQ ID Nos. 10–18 or 20, though sequences identical tothose sequence listing entries are also contemplated as useful in thepresent method. The hedgehog portion can be encoded by a nucleic acidwhich hybridizes under stringent conditions to a nucleic acid sequenceof any of SEQ ID Nos. 1–9 or 19, e.g., the hedgehog portion can beencoded by a vertebrate hedgehog gene, especially a human hedgehog gene.

In certain embodiments, the hedgehog proteins of the present inventionare modified by a lipophilic moiety or moieties at one or more intenalsites of the mature, processed extracellular domain, and may or may notbe also derivatized with lipophilic moieties at the N or C-terminalresidues of the mature polypeptide. In other embodiments, thepolypeptide is modified at the C-terminal residue with a hydrophobicmoiety other than a sterol. In still other embodiments, the polypeptideis modified at the N-terminal residue with a cyclic (preferablypolycyclic) lipophilic group. Various combinations of the above are alsocontemplated.

In other embodiments, the subject method can be carried out byadministering a gene activation construct, wherein the gene activationconstruct is designed to recombine with a genomic hedgehog gene of thepatient to provide a heterologous transcriptional regulatory sequenceoperatively linked to a coding sequence of the hedgehog gene.

In still other embodiments, the subject method can be practiced with theadministration of a gene therapy construct encoding a hedgehogpolypeptide. For instance, the gene therapy construct can be provided ina composition selected from a group consisting of a recombinant viralparticle, a liposome, and a poly-cationic nucleic acid binding agent.

Another aspect of the present invention relates to the cloning ofvarious human hedgehog genes, e.g., human Dhh and Ihh. In a preferredembodiment, there is provided an isolated and/or recombinantly producedpolypeptide comprising an amino acid sequence which is at least 95percent identical to a sequence represented by SEQ ID. NO. 16 or 17, ora bioactive extracellular fragment thereof. In another embodiment, thereis provided an isolated and/or recombinantly produced polypeptideencoded by a nucleic acid which hybridizes under stringent conditions toa sequence selected from the group consisting of SEQ ID. NO. 16 and SEQID. NO. 17. In a preferred embodiment, the polypeptide is formulated ina pharmaceutically acceptable carrier.

Preferred bioactive fragments of the human Ihh and Dhh proteins includefrom about residues 28–202 of SEQ ID No. 16 and 23–198 of SEQ ID No. 17,respectively. Longer or shorter fragments are contemplated, as forexample, those which are 5, 10, 15 or 20 amino acids shorter on eitheror both the N-terminal and C-terminal ends of the fragment.

In certain embodiments, the polypeptide is purified to at least 80% bydry weight, and more preferably 90 or 95% by dry weight.

Another aspect of the present invention provides an isolated nucleicacid encoding a polypeptide comprising a hedgehog amino acid sequencewhich is at least 95 percent identical to a hedgehog protein selectedfrom the group consisting of SEQ ID No:16 and SEQ ID No:17, or bioactivefragments thereof, e.g., the hedgehog amino acid sequence (i) binds to apatched protein, (ii) regulates differentiation of neuronal cells, (iii)regulates survival of differentiated neuronal cells, (iv) regulatesproliferation of chondrocytes, (v) regulates proliferation of testiculargerm line cells, or (vi) functionally replaces drosopholia hedgehog intransgenic drosophila fly, or a combination thereof.

In other preferred embodiments, the isolated nucleic acid encodes apolypeptide having a hedgehog amino acid sequence encoded by a nucleicacid which hybridizes under stringent conditions to a nucleic acidsequence selected from the group consisting of SEQ ID No:7 and SEQ IDNo:8, which hedgehog amino acid sequence of the polypeptide correspondsto a natural proteolytic product of a hedgehog protein. Suchpolypeptides preferably (i) binds to a patched protein, (ii) regulatesdifferentiation of neuronal cells, (iii) regulates survival ofdifferentiated neuronal cells, (iv) regulates proliferation ofchondrocytes, (v) regulates proliferation of testicular germ line cells,and/or (vi) functionally replaces drosophila hedgehog in transgenicdrosophila fly, or a combination thereof.

In preferred embodiments, the nucleic acid encodes a hedgehog amino acidsequence identical to a hedgehog protein selected from the groupconsisting of SEQ ID No:16 and SEQ ID No:17.

Another preferred ebodiment provides an isolated nucleic acid comprisinga coding sequence of a human hedgehog gene, encoding a bioactivehedgehog protein.

Still another aspect of the present invention relates to an expressionvector, capable of replicating in at least one of a prokaryotic cell andeukaryotic cell, comprising a nucleic acid encoding a Dhh or Ihhpolypeptide described above.

The present invention also provides a host cell transfected with suchexpression vectors; as well as methods for producing a recombinanthedgehog polypeptide by culturing such cells in a cell culture medium toexpress a hedgehog polypeptide and isolating said hedgehog polypeptidefrom the cell culture.

Still another aspect of the present invention provides a recombinanttransfection system, e.g., such as may be useful for gene therapy,comprising (i) a gene construct including the coding sequence for ahuman Ihh or Dhh protein, operably linked to a transcriptionalregulatory sequence for causing expression of the hedgehog polypeptidein eukaryotic cells, and (ii) a gene delivery composition for deliveringsaid gene construct to a cell and causing the cell to be transfectedwith said gene construct. For instance, the gene delivery composition isselected from a group consisting of a recombinant viral particle, aliposome, and a poly-cationic nucleic acid binding agent.

Another aspect of the present invention provides a probe/primercomprising a substantially purified oligonucleotide, saidoligonucleotide containing a region of nucleotide sequence whichhybridizes under stringent conditions to at least 10 consecutivenucleotides of sense or antisense sequence of SEQ ID No. 7 or 8, ornaturally occuring mutants thereof. In preferred embodiments, theprobe/primer includes a label group attached thereto and able to bedetected. The present invention also provides a test kit for detectingcells which contain a hedgehog mRNA transcript, and includes suchprobe/primers.

Still another embodiment of the present invention provides a purifiedpreparation of an antisense nucleic acid which specifically hybridizesto and inhibits expression of a gene encoding a human Ihh or Dhhhedgehog protein under physiological conditions, which nucleic acid isat least one of (i) a synthetic oligonucleotide, (ii) single-stranded,(iii) linear, (iv) 20 to 50 nucleotides in length, and (v) a DNA analogresistant to nuclease degradation.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Shh and Ptc in the E14.5 rat embryo. Shh (A, antisense; B, sensecontrol), and ptc (C, antisense; D, sense control) expression asdetected by in situ hybridization with digoxigenin-labeled riboprobesand alkaline phosphatase-conjugated anti-digoxigenin. The arrow in A andthe double-arrow in C designate the zona limitan intrathalamica. Majoranatomical structures and summary diagrams of shh and ptc expression areshown in E. Scale bar=1 mm.

FIG. 2. Shh promotes the survival of TH+ neurons of the ventralmesencephalon. (A) Timecourse and dose response of the Shh effect. Thenumber of TH+ neurons in control cultures (0 ng/ml Shh) began to declinedramatically by 5 days in vitro. In cultures treated with Shh at 25 and50 ng/ml there were significantly greater numbers of TH+ nurrons overcontrol-through 24 days in vitro (from 5 to 24 days, p<0.001 at 25 and50 ng/ml). The 50 ng/ml dose typically gave a 50–100% increase overcontrols at all time points (error bars s.e.m.)P-h'otomicrographs of TH+neurons in 50 .ng/ml Shh treated (B-,,,-D) and control (C, E) cultures,2 days (B, C) and 7 days (D, E) post-plating. Note that in addition toan increased number of TH+-cell bodies, the Shh treated cells—showextensive neuritic processes. Scale bar=200 um.

FIG. 3. Transport of 3H-Dopamine. The identity and functionality of thesurviving midbrain neurons was assessed by their ability to specificallytransport dopamine. (A) Addition of 25 ng/ml Shh resulted in a 22-foldincrease in 3H-DA cell uptake over controls and lower Shhconcentrations. 50 ng/ml Shh gave a 30-fold increase in 3H-DA uptake(error bars=s.d.) (p<0.005 at 25 and 50 ng/ml). (B) Autoradiography wasperformed on sister plates to visualize dopamine transport. Only cellswith neuronal morphology transported 3H-DA (inset). Scale bar=50 Jim,inset 15 m.

FIG. 4. Specificity of Shh activity. (A) QC-PCR gel. Lanes 1–4 are cDNAfrom midbrain cultures that have been co-amplified with successive4-fold dilutions of mimic oligo. Lane 5 is DNA marker lane. Ptc targetis 254 bp and mimic is 100 bp(B) Representative plot (corresponding toA) of the log concentration of competitive mimic versus the log of theobtained band densities of target and mimic PCR substrates demonstratesthe linearity of the amplification reaction. The extrapolated value ofptc message in the cDNA tested is determined to be equal to the value ofmimic concentration where Log Ds/Dm=0. See main text for details of theprocedure. Doses in ng/ml; Ds=density of test substrate; Dm=density ofcompetitive mimic. The r 2 value shows that determinations made withinthis range vary within 3%. (C) Administration of Shh induces ptcexpression in a dose response that parallels the survival curve. Thevalues are expressed as number of target molecules (log Ds) per totalamount of cDNA used in each reaction as measured by optical density at260 nm (OD) and were determined as demonstrated in A and B. At 4 days invitro Shh at 5 ng/ml increases ptc expression over control, and 50 ng/mlincreases expression of ptc over the level found in the ventralmesencephalon at the time of dissection. (D) Affinity purified anti-Shhantibody inhibited the Shh neurotrophic response (p<0.001). Cultureswere maintained for 5 days. Shh was added at a concentration of 50ng/ml, and in the co-administration of 5 Shh and anti-Shh (“Shhantibody”) Shh-was added at 0 g/ml and anti-Shh was added as a 5-foldmolar excess (error bars s.e.m.).

FIG. 5. Shh also supports the survival of midbrain-GABA+ neurons. (A) Inaddition to supporting the survival of TH+ cells in the midbraincultures, Shh promotes the survival of GABA-immunoreactive neurons witha similar dose response (error bars=s.e.m.) (For TH, p, 0.001 at 25 and50 ng/ml; for GABA, p<0.001 at 25 and 50 ng/ml). (B) Double levelimmunofluorescence of SSH-treated cultures shows that the majority ofthe GABA+ cells (Orange) do not overlap with the TH+ cells (green);scale bar=15 m.

FIG. 6. Shh effects on striatal cultures. (A) At concentrations of 10ng/ml and higher, Shh promotes neuronal survival as gauged by stainingfor tubulin PIII, and these cells are exclusively GABA+ (errorbars=S.D.) (tubulin PIII, p<0.001 at 25 and 50 ng/ml; GABA, p<0.001 at25 and 50 ng/ml). Typical fields of neurons treated with 50 ng/ml Shhstained for tubulin pIII (B) and GABA+ (C) are shown; scale bar=100 gm.

FIG. 7. Shh effects on ventral spinal cultures. (A) At concentrations of25 ng/ml and higher, Shh promotes neuronal survival as gauged bystaining for tubulin PIII. The majority of the cells stain positivelyfor GABA, while a subset stain for the nuclear marker of spinalinterneurons, Lim-1/2 (error bars=s.e.m.) (tubulin pill, p<0.001 at 25and 50 ng/ml; lim 1/2, p<0.001 at 5, 10, 25, and 50 ng/ml; GABA, p<0.001at 25 and 50 ng/ml). Typical staining for Lim-−1/2 in the E14 rat spinalcord (B, scale bar=100 m), and spinal neurons cultured in the presenceof 50 ng/ml Shh (C, scale bar=20 m).

FIG. 8. Shh protects midbrain TH+ neurons from neurotoxic insult.Cultures of ventral mesencephalon neurons were cultured in the indicatedconcentrations of Shh (ng/ml). MPP+ was added at 4 days in vitro for 48hours. Cultures were then washed extensively and cultured for anadditional 48 hours to allow clearance of dying neurons. Protection fromMPP+ neurotoxicity could be seen at 5 ng/ml, with the effect saturatingat 50 ng/ml. BDNF was used at 10 ng/ml, and GDNF at 20 ng/ml (error barss.e.m.) (Shh, p<0.001 at 50 and 250 ng/ml; BDNF no significance; GDNF,p<0.05). Note that the plating density used in this experiment was twicethat used in FIG. 2.

DETAILED DESCRIPTION OF THE INVENTION

Sonic hedgehog (Shh), an axis-determining secreted protein, is expressedduring early vertebrate embryogenesis in the notochord and ventralneural tube. In this site it plays a role in the phenotypicspecification of ventral neurons along the length of the CNS. Forexample, Shh induces the differentiation of motor neurons in the spinalcord and dopaminergic neurons in the midbrain. Shh expression, however,persists beyond this induction period. We have show here that Shhpossesses novel activities beyond phenotype specification. Usingcultures derived from the embryonic day 14.5 (E14.5) rat ventralmesencephalon, we show that Shh is also trophic for dopaminergicneurons. Interestingly, Shh not only promotes dopaminergic neuronsurvival, but also promotes the survival of midbrain GABA-immunoeractive(GABA-ir) neurons. In cultures derived from the E15–16 striatum, Shhpromotes the survival of GABA-ir interneurons to the exclusion of anyother cell type. Cultures derived from E15–16 ventral spinal cord revealthat Shh is again trophic for interneurons, many of which are GABA-irand some of which express the Lim-1/2 nuclear marker, but does notappear to support motorneuron survival. Shh does not support survival ofsympathetic or dorsal root ganglion neurons. Finally, using the midbraincultures, we show that in the presence of MPP+, a highly specificneurotoxin, Shh prevents dopaminergic neuron death that normally wouldhave occurred.

Based in part on these findings, we have determined that Shh, and otherforms of hedgehog proteins, are useful as a protective agents in thetreatment and prophylaxis for neurodegenerative disorders, particularlythose resulting from the loss of dopaminergic and/or GABA-nergicneurons, or the general loss tissue from the substantia nigra. Asdescribed with greater detail below, exemplary disorders (“candidatedisorders”) include Parkinson's disease, Huntington's disease,amyotrophic lateral sclerosis and the like.

The subject invention also utilizies hedgehog or hedgehog agonists ascell culture additives for the maintenance of differentiated neurons incultures, e.g., in cultures of dopaminergic and GABA-nergic neurons. Thesubject methods and compositions can also be used to augment theimplantion of such neuronal cells in an animal.

In terms of treatment, once a patient experiences symptoms of acandidate disorder, a goal of therapy is prevention of further loss ofneuron function.

I. Overview

The present application is directed to compositions and methods for theprevention and treatment of ischemic injury to the brain, such asresulting from stroke. The invention derives, at least in part, from theobservation of a protective effect by the so called “hedgehog” proteinson animal stroke models. Briefly, as described in the appended examples,we investigated the neuroprotective potential of hedgehog proteins in arat model of focal cerebral ischemia that used permanent occlusion ofthe middle cerebral artery. Intravenous infusion of vehicle (control) orShh (sonic hedgehog) was administered for 3 hours beginning 30 minutesafter occlusion, and resulted in a 70 percent reduction in total infarctsize (P=0.0039), relative to the control, when examined 24 hourspost-occlusion. Measurements of arterial blood pressure, blood gases,glucose, hematocrit and osmolality revealed no difference among vehicle-and Shh-treated animals. These results show that the intravenoushedgehog protein reduces neuronal damage due to stroke.

In one aspect, the present invention provides pharmaceuticalpreparations and methods for preventing/treating cerebral ischemia andthe like utilizing, as an active ingredient, a hedgehog polypeptide or amimetic thereof.

The subject hedgehog treatments are effective on both human and animalsubjects afflicted with these conditions. Animal subjects to which theinvention is applicable extend to both domestic animals and livestock,raised either as pets or for commercial purposes. Examples are dogs,cats, cattle, horses, sheep, hogs and goats.

However, without wishing to be bound by any particular theory, thereduction in infarct size in the present studies may be due at least inpart to the ability of hedgehog proteins to antagonize (directly orindirectly) patched-mediated regulation of gene expression and otherphysiological effects mediated by the patched gene. The patched geneproduct, a cell surface protein, is understood to signal through apathway which regulates transcription of a variety of genes involved inneuronal cell development. In the CNS and other tissue, the introductionof hedgehog relieves (derepresses) this inhibition conferred by patched,allowing expression of particular gene programs.

Accordingly, the present invention contemplates the use of other agentswhich are capable of mimicking the effect of the hedgehog protein onpatched signalling, e.g., as may be identified from the drug screeningassays described below.

II. Definitions

For convenience, certain terms employed in the specfication, examples,and appended claims are collected here.

The term “hedgehog therapeutic” refers to various forms of hedgehogpolypeptides, as well as peptidomimetics, which are neuroprotective forneuronal cells, and in particular, enhance the survival of dopaminergicand GABA-ergic neurons. These include naturally occurring forms ofhedgehog proteins, as well as modified or mutant forms generated bymolecular biological techniques, chemical synthesis, etc. While inpreferred embodiments the hedgehog polypeptide is derived from avertebrate homolog, cross-sepcies activity reported in the literaturesupports the use of hedgehog peolypeptides from invertebrate organismsas well. Naturally and non-naturally occurring hedgehog therapeuticsreferred to herein as “agonists” mimic or potentiate (collectively“agonize”) the effects of a naturally occurring hedgehog protein as aneuroprotective agent. In addition, the term “hedgehog therapeutic”includes molecules which can activate expression of an endogenoushedgehog gene. The term also includes gene therapy constructs forcausing expression of hedgehog polypeptides in vivo, as for example,expression constructs encoding recombinant hedgehog polypeptides as wellas trans-activation constructs for altering the regulatory sequences ofan endogenous hedgehog gene by homologous recombination.

In particular, the term “hedgehog polypeptide” encompasses hedgehogproteins and peptidyl fragments thereof.

As used herein the term “bioactive fragment”, with reference to aportions of hedgehog proteins, refers to a fragment of a full-lengthhedgehog protein, wherein the fragment specifically agonizesneuroprotective events mediated by wild-type hedgehog proteins. Thehedgehog bioactive fragment preferably is a soluble extracellularportion of a hedgehog protein, where solubility is with reference tophysiologically compatible solutions. Exemplary bioactive fragments aredescribed in PCT publications WO 95/18856 and WO 96/17924.

The term “ptc therapeutic” refers to agents which mimic the effect ofnaturally occurring hedgehog proteins on patched signalling. The ptctherapeutic can be, e.g., a peptide, a nucleic acid, a carbohydrate, asmall organic molecule, or natural product extract (or fractionthereof).

A “patient” or “subject” to be treated by the subject method is amammal, including a human.

An “effective amount” of, e.g., a hedgehog or ptc therapeutic, withrespect to the subject method of treatment, refers to an amount of thetherapeutic in a preparation which, when applied as part of a desireddosage regimen causes a increase in survival of a neuronal cellpopulation according to clinically acceptable standards for thetreatment or prevention of a particular disorder.

By “prevent degeneration” it is meant reduction in the loss of cells(such as from apoptosis), or reduction in impairment of cell function,e.g., release of dopamine in the case of dopaminergic neurons.

A “trophic factor”, referring to a hedgehog or ptc therapeutic, is amolecule that directly or indirectly affects the survival or function ofa hedgehog-responsive cell, e.g., a dopaminergic or GABAergic cell.

A “trophic amount” of a a hedgehog or ptc therapeutic is an amountsufficient to, under the circumstances, cause an increase in the rate ofsurvival or the functional perfomance of a hedgehog-responsive cell,e.g., a dopaminergic or GABAergic cell.

“Homology” and “identity” each refer to sequence similarity between twopolypeptide sequences, with identity being a more strict comparison.Homology and identity can each be determined by comparing a position ineach sequence which may be aligned for purposes of comparison. When aposition in the compared sequence is occupied by the same amino acidresidue, then the polypeptides can be referred to as identical at thatposition; when the equivalent site is occupied by the same amino acid(e.g., identical) or a similar amino acid (e.g., similar in stericand/or electronic nature), then the molecules can be referred to ashomologous at that position. A percentage of homology or identitybetween sequences is a function of the number of matching or homologouspositions shared by the sequences. An “unrelated” or “non-homologous”sequence shares less than 40 percent identity, though preferably lessthan 25 percent identity, with an AR sequence of the present invention.

The term “corresponds to”, when referring to a particular polypeptide ornucleic acid sequence is meant to indicate that the sequence of interestis identical or homologous to the reference sequence to which it is saidto correspond.

The terms “recombinant protein”, “heterologous protein” and “exogenousprotein” are used interchangeably throughout the specification and referto a polypeptide which is produced by recombinant DNA techniques,wherein generally, DNA encoding the polypeptide is inserted into asuitable expression construct which is in turn used to transform a hostcell to produce the heterologous protein. That is, the polypeptide isexpressed from a heterologous nucleic acid.

A “chimeric protein” or “fusion protein” is a fusion of a first aminoacid sequence encoding a hedgehog polypeptide with a second amino acidsequence defining a domain foreign to and not substantially homologouswith any domain of hh protein. A chimeric protein may present a foreigndomain which is found (albeit in a different protein) in an organismwhich also expresses the first protein, or it may be an “interspecies”,“intergenic”, etc. fusion of protein structures expressed by differentkinds of organisms. In general, a fusion protein can be represented bythe general formula (X)_(n)-(hh)_(m)-(Y)_(n), wherein hh represents allor a portion of the hedgehog protein, X and Y each independentlyrepresent an amino acid sequences which are not naturally found as apolypeptide chain contiguous with the hedgehog sequence, m is an integergreater than or equal to 1, and each occurrence of n is, independently,0 or an integer greater than or equal to 1 (n and m are preferably nogreater than 5 or 10).

As used herein, the term “vector” refers to a nucleic acid moleculecapable of transporting another nucleic acid to which it has beenlinked. The term “expression vector” includes plasmids, cosmids orphages capable of synthesizing, for example, the subject hedgehogpolypeptides encoded by the respective recombinant gene carried by thevector. Preferred vectors are those capable of autonomous replicationand/expression of nucleic acids to which they are linked. In the presentspecification, “plasmid” and “vector” are used interchangeably as theplasmid is the most commonly used form of vector. Moreover, theinvention is intended to include such other forms of expression vectorswhich serve equivalent functions and which become known in the artsubsequently hereto.

“Transcriptional regulatory sequence” is a generic term used throughoutthe specification to refer to DNA sequences, such as initiation signals,enhancers, and promoters, as well as polyadenylation sites, which induceor control transcription of protein (or antisense) coding sequences withwhich they are operably linked. In preferred embodiments, transcriptionof a recombinant gene is under the control of a promoter sequence (orother transcriptional regulatory sequence) which controls the expressionof the recombinant gene in a cell-type in which expression is intended.It will also be understood that the recombinant gene can be under thecontrol of transcriptional regulatory sequences which are the same orwhich are different from those sequences which control transcription ofthe naturally-occurring form of the regulatory protein.

The term “operably linked” refers to the arrangement of atranscriptional regulatory element relative to another transcribablenucleic acid sequence, such that the transcriptional regulatory elementcan regulate the rate of transcription from the transcribablesequence(s).

III. Exemplary Applications of Method and Compositions

One aspect of the present invention relates to a method of maintaining adifferentiated state, e.g., enhancing survival, of a neuronal cellresponsive to a hedgehog protein, by contacting the cells with a trophicamount of a hedgehog or ptc thereapeutic. For instance, it iscontemplated by the invention that, in light of the present finding ofan apparently trophic effect of hedgehog proteins in the maintenance ofdifferentiated neurons, the subject method could be used to maintaindifferent neuronal tissue both in vitro and in vivo. Where the trophicagent is a hedgehog protein, it can be provided to a cell culture oranimal as a purified protein or secreted by a recombinant cell, or cellsor tissue explants which naturally produce one or more hedgehogproteins. For instance, neural tube explants from embryos, particularlyDoorplate tissue, can provide a source for Shh polypeptide, which sourcecan be implanted in a patient or otherwise provided, as appropriate, formaintenance of differentiation.

The present method is applicable to cell culture techniques. In vitroneuronal culture systems have proved to be fundamental and indispensabletools for the study of neural development, as well as the identificationof neurotrophic factors such as nerve growth factor (NGF), ciliarytrophic factors (CNTF), and brain derived neurotrophic factor (BDNF).Once a neuronal cell has become terminally-differentiated it typicallywill not change to another terminally differentiated cell-type. However,neuronal cells can nevertheless readily lose their differentiated state.This is commonly observed when they are grown in culture from adulttissue, and when they form a blastema during regeneration. The presentmethod provides a means for ensuring an adequately restrictiveenvironment in order to maintain dopaminergic and GABAergic cells indifferentiated states, and can be employed, for instance, in cellcultures designed to test the specific activities of other trophicfactors.

In such embodiments of the subject method, a culture of differentiatedcells inlcuding dopaminergic and/or GABAergic cells can be contactedwith a hedgehog or ptc therapeutic in order to maintain the integrity ofa culture of terminally-differentiated neuronal cells by preventing lossof differentiation. The source of hedgehog or ptc therapeutic in theculture can be derived from, for example, a purified or semi-purifiedprotein composition added directly to the cell culture media, oralternatively, supported and/or released from a polymeric device whichsupports the growth of various neuronal cells and which has been dopedwith the protein. The source of, for example, a trophic hedgehogpolypeptide can also be a cell that is co-cultured with the neuronalcells. Alternatively, the source can be the neuronal cell itself whichhas been engineered to produce a recombinant hedgehog protein. Suchneuronal cultures can be used as convenient assay systems as well assources of implantable cells for therapeutic treatments.

The subject method can be used in conjunction with agents which inducethe differentiation of neuronal precursors, e.g., progenitor or stemcells, into dopaminergic or GABAergic neurons.

Cells can be obtained from embryonic, post-natal, juvenile or adultneural tissue from any animal. By any animal is meant any multicellularanimal which contains nervous tissue. More particularly, is meant anyfish, reptile, bird, amphibian or mammal and the like. The mostpreferable donors are mammals, especially humans and non-human primates,pigs, cows, and rodents.

Intracerebral neural grafting has emerged recently as an additionalpotential to CNS therapy. For example, one approach to repairing damagedbrain tissues involves the transplantation of cells from fetal orneonatal animals into the adult brain (Dunnett et al. (1987) J Exp Biol123:265–289; and Freund et al. (1985) J Neurosci 5:603–616). Fetalneurons from a variety of brain regions can be successfully incorporatedinto the adult brain, and such grafts can alleviate behavioral defects.For example, movement disorder induced by lesions of dopaminergicprojections to the basal ganglia can be prevented by grafts of embryonicdopaminergic neurons. Complex cognitive functions that are impairedafter lesions of the neocortex can also be partially restored by graftsof embryonic cortical cells. Transplantation of fetal brain cells, whichcontain precursors of the dopaminergic neurons, has been examined withsuccess as a treatment for Parkinson's disease. In animal models and inpatients with this disease, fetal brain cell transplantations haveresulted in the reduction of motor abnormalities. Furthermore, itappears that the implanted fetal dopaminergic neurons form synapses withsurrounding host neurons. However, in the art, the transplantation offetal brain cells is limited due, for example, to the limited survivaltime of the implanted neuronal precursors and differentiated neuronsarising therefrom. The subject invention provides a means for extendingthe usefulness of such transplants by enhancing the survival ofdopaminergic and/or GABAergic cells in the transplant.

In the specific case of Parkinson's disease, intervention by increasingthe activity of hedgehog, by ectopic or endogenous means, can improvethe in vivo survival of fetal and adult dopaminergic neurons, and thuscan provide a more effective treatment of this disease. Cells to betransplanted for the treatment of a particular disease can begenetically modified in vitro so as to increase the expression ofhedgehog in the transplant. In an exemplary embodiment of the invention,administration of an Shh polypeptide can be used in conjunction withsurgical implantation of tissue in the treatment of Parkinson's disease.

In the case of a heterologous donor animal, the animal may beeuthanized, and the brain and specific area of interest removed using asterile procedure. Brain areas of particular interest include any areafrom which progenitor cells can be obtained which will providedopaminergic or GABAergic cells upon differentiation. These regionsinclude areas of the central nervous system (CNS) including thesubstantia nigra pars compacta which is found to be degenerated inParkinson's Disease patients.

Human heterologous neural progenitor cells may be derived from fetaltissue obtained from elective abortion, or from a post-natal, juvenileor adult organ donor. Autologous neural tissue can be obtained bybiopsy, or from patients undergoing neurosurgery in which neural tissueis removed, such as during epilepsy surgery.

Cells can be obtained from donor tissue by dissociation of individualcells from the connecting extracellular matrix of the tissue.Dissociation can be obtained using any known procedure, includingtreatment with enzymes such as trypsin, collagenase and the like, or byusing physical methods of dissociation such as with a blunt instrument.Dissociation of fetal cells can be carried out in tissue culture medium,while a preferable medium for dissociation of juvenile and adult cellsis artificial cerebral spinal fluid (aCSF). Regular aCSF contains 124 mMNaCl, 5 mM KCl, 1.3 mM MgCl₂, 2 mM CaCl₂, 26 mM NaHCO₃, and 10 mMD-glucose. Low Ca²⁺ aCSF contains the same ingredients except for MgCl₂at a concentration of 3.2 mM and CaCl₂ at a concentration of 0.1 mM.

Dissociated cells can be placed into any known culture medium capable ofsupporting cell growth, including MEM, DMEM, RPMI, F-12, and the like,containing supplements which are required for cellular metabolism suchas glutamine and other amino acids, vitamins, minerals and usefulproteins such as transferrin and the like. Medium may also containantibiotics to prevent contamination with yeast, bacteria and fungi suchas penicillin, streptomycin, gentamicin and the like. In some cases, themedium may contain serum derived from bovine, equine, chicken and thelike. A particularly preferable medium for cells is a mixture of DMEMand F-12.

Conditions for culturing should be close to physiological conditions.The pH of the culture media should be close to physiological pH,preferably between pH 6–8, more preferably close to pH 7, even moreparticularly about pH 7.4. Cells should be cultured at a temperatureclose to physiological temperature, preferably between 30° C.–40° C.,more preferably between 32° C.–38° C., and most preferably between 35°C.–37° C.

Cells can be grown in suspension or on a fixed substrate, butproliferation of the progenitors is preferably done in suspension togenerate large numbers of cells by formation of “neurospheres” (see, forexample, Reynolds et al. (1992) Science 255:1070–1709; and PCTPublications WO93/01275, WO94/09119, WO94/10292, and WO94/16718). In thecase of propagating (or splitting) suspension cells, flasks are shakenwell and the neurospheres allowed to settle on the bottom corner of theflask. The spheres are then transferred to a 50 ml centrifuge tube andcentrifuged at low speed. The medium is aspirated, the cells resuspendedin a small amount of medium with growth factor, and the cellsmechanically dissociated and resuspended in separate aliquots of media.

Cell suspensions in culture medium are supplemented with any growthfactor which allows for the proliferation of progenitor cells and seededin any receptacle capable of sustaining cells, though as set out above,preferably in culture flasks or roller bottles. Cells typicallyproliferate within 3–4 days in a 37° C. incubator, and proliferation canbe reinitiated at any time after that by dissociation of the cells andresuspension in fresh medium containing growth factors.

In the absence of substrate, cells lift off the floor of the flask andcontinue to proliferate in suspension forming a hollow sphere ofundifferentiated cells. After approximately 3–10 days in vitro, theproliferating clusters (neurospheres) are fed every 2–7 days, and moreparticularly every 2–4 days by gentle centrifugation and resuspension inmedium containing growth factor.

After 6–7 days in vitro, individual cells in the neurospheres can beseparated by physical dissociation of the neurospheres with a bluntinstrument, more particularly by triturating the neurospheres with apipette. Single cells from the dissociated neurospheres are suspended inculture medium containing growth factors, and differentiation of thecells can be induced by plating (or resuspending) the cells in thepresence of a factor capable of sustaining differentiation, e.g., suchas a hedgehog or ptc therapeutic of the present invention.

Stem cells useful in the present invention are generally known. Forexample, several neural crest cells have been identified, some of whichare multipotent and likely represent uncommitted neural crest cells. Therole of hedgehog proteins employed in the present method to culture suchstem cells is to maintain differentiation a committed progenitor cellamd/or a terminally-differentiated dopaminergic or GABAergic neuronalcell. The hedgehog protein can be used alone, or can be used incombination with other neurotrophic factors which act to moreparticularly enhance a particular differentiation fate of the neuronalprogenitor cell.

In addition to the implantation of cells cultured in the presence of afunctional hedgehog activity and other in vitro uses described above,yet another aspect of the present invention concerns the therapeuticapplication of a hedgehog or ptc therapeutic to enhance survival ofdopaminergic and GABAergic neurons in vivo. The ability of hedgehogprotein to maintain dopaminergic and GABAergic neuronal differentiationindicates that certain of the hedgehog proteins can be reasonablyexpected to facilitate control of of these neuronal cell-types in adulttissue with regard to maintenance, functional performance, aging andprevention of degeneration and premature death which result from loss ofdifferentiation in certain pathological conditions. In light of thisunderstanding, the present invention specifically contemplatesapplications of the subject method to the treatment of (preventionand/or reduction of the severity of) neurological conditions derivingfrom (i) loss of dopaminergic cells, (ii) loss of GABAergic cells,and/or (iii) loss of neurons of the substantia nigra. In this regard,the subject method is useful in the treatment of chronicneurodegenerative diseases of the nervous system, including Parkinson'sdisease, Huntington's chorea, amylotrophic lateral sclerosis and thelike.

Many neurological disorders are associated with degeneration of discretepopulations of neuronal elements and may be treatable with a therapeuticregimen which includes a hedgehog or ptc therapeutic according to thesubject invention. As described in the appended examples, hedgehogexerts trophic and survival-promoting actions on substantia nigradopaminergic neurons. In vivo, treatment with exogenous hedgehog, orother compounds of the present invention, is expected to stimulate thedopaminergic phenotype of substantia nigra neurons and restoresfunctional deficits induced by axotomy or dopaminergic neurotoxins, andmay be used the treatment of Parkinson's disease, a neurodegenerativedisease characterized by the loss of dopaminergic neurons. Thus, in oneembodiment, the subject method comprises administering to an animalafflected with Parkinson's disease, or at risk of developing Parkinson'sdisease, an amount of a hedgehog or ptc thereapeutic effective forincreasing the rate of survival of dopaminergic neurons in the animal.In preferred embodiments, the method includes administering to theanimal an amount of a hedgehog or ptc thereapeutic which would otherwisebe effective at protecting the substantia nigra from MPTP-mediatedtoxicity when MPTP is administered at a dose of 0.5 mg/kg, morepreferably at a dose of 2 mg/kg, 5 mg/kg, 10 mg/kg, 20 mg/kg or 50 mg/kgand, more preferably, at a dose of 100 mg/kg.

Huntington's disease involves the degeneration of intrastraital andcortical cholinergic neurons and GABAergic neurons. Treatment ofpatients suffering from such degenerative conditions can include theapplication of hedgehog or ptc therapeutics of the present invention, inorder to control, for example, apoptotic events which give rise to lossof GABAergic neurons (e.g. to enhance survival of existing neurons.

Recently it has been reported that in certain ALS patients and animalmodels a significant loss of midbrain dopaminergic neurons occurs inaddition to the loss of spinal motor neurons. For instance, theliterature describes degeneration of the substantia nigra in somepatients with familial amyotrophic lateral sclerosis. Kostic et al.(1997) Ann Neurol 41:497–504. According the subject invention, a trophicamount of a hedgehog or ptc therapeutic can be administered to an animalsuffering from, or at risk of developing, ALS.

In general, the therapeutic method of the present invention can becharacterized as including a step of administering to an animal anamount of a ptc or hedgehog therapeutic effective to enhance thesurvival of a dopaminergic and/or GABAergic neuronal cells. The mode ofadministration and dosage regimens will vary depending on the severityof the degenerative disoder being treated, e.g., the dosage may bealtered as between a prophylaxis and treatment. In preferredembodiments, the ptc or hedeghog therapeutic is administeredsystemically initially, then locally for medium- to long-term care. Incertain embodiments, a source of a hedgehog or ptc therapeutic isstereotactically provided within or proximate the area of degeneration.

The subject method may also find particular utility in treating orpreventing the adverse neurological consequences of surgery. Forexample, certain cranial surgery can result in degeneration of neuronalpopulations for which the subject method can be applied.

In other embodiments, the subject method can be used to prevent or treatneurodegenerative conditions arising from the use of certain drugs, suchas the compound MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine).

In still other embodiments, the subject method can be used in theprevention and/or treatment of hypoxia, e.g., as a neuroprotectiveagent. For instance, the subject method can be used prophylactically tolessen the neuronal cell death caused by altitude-induced hypoxia.

A method which is “neuroprotective”, in the case of dopaminergic andGABAergic cells, results in diminished loss of cells of those phenotyperelative to that which would occur in the absence of treatment with ahedgehog or ptc therapeutic.

In yet other embodiments, the subject method can be carried outconjointly with the administration of growth and/or trophic factors. Forinstance, the combinatorial therapy can include a trophic factor such asnerve growth factor, cilliary neurotrophic growth factor,schwanoma-derived growth factor, glial growth factor, stiatal-derivedneuronotrophic factor, platelet-derived growth factor, and scatterfactor (HGF-SF). Antimitogenic agents can also be used, as for example,cytosine, arabinoside, 5-fluorouracil, hydroxyurea, and methotrexate.

Determination of a therapeutically effective amount and aprophylactically effective amount of a hedgehog or ptc therapeutic,e.g., to be adequately neuroprotective, can be readily made by thephysician or veterinarian (the “attending clinician”), as one skilled inthe art, by the use of known techniques and by observing resultsobtained under analogous circumstances. The dosages may be varieddepending upon the requirements of the patient in the judgment of theattending clinician, the severity of the condition being treated, therisk of further degeneration to the CNS, and the particular agent beingemployed. In determining the therapeutically effective trophic amount ordose, and the prophylactically effective amount or dose, a number offactors are considered by the attending clinician, including, but notlimited to: the specific cause of the degenerative state and itslikelihood of recurring or worsening; pharmacodynamic characteristics ofthe particular agent and its mode and route of administration; thedesired time course of treatment; the species of mammal; its size, age,and general health; the response of the individual patient; theparticular compound administered; the bioavailability characteristics ofthe preparation administered; the dose regimen selected; the kind ofconcurrent treatment (i.e., the interaction of the hedgehog or ptctherapeutic with other co-administered therapeutics); and other relevantcircumstances.

Treatment can be initiated with smaller dosages which are less than theoptimum dose of the agent. Thereafter, the dosage should be increased bysmall increments until the optimum effect under the circumstances isreached. For convenience, the total daily dosage may be divided andadministered in portions during the day if desired. A therapeuticallyeffective trophic amount and a prophylactically effectiveneuroprotective amount of a hedgehog polypeptide, for instance, isexpected to vary from concentrations about 0.1 nanogram per kilogram ofbody weight per day (ng/kg/day) to about 100 mg/kg/day.

Potential hedgehog and ptc therapeutics, such as described below, can betested by any of number of well known animal disease models. Forinstance, regarding Parkinson's Disease, selected agents can beevaluated in animals treated with MPTP. The compound MPTP(1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) and its metabolite MPP⁺have been used to induce experimental parkinsonism. MPP⁺ killsdopaminergic neurons in the substantia nigra, yielding a reasonablemodel of late parkinsonism. Turski et al., (1991) Nature 349:414.

Compounds which are determined to be effective for the prevention ortreatment of degeneration of dopaminergic and GABAergic neurons and thelike in animals, e.g., dogs, rodents, may also be useful in treatment ofdisorders in humans. Those skilled in the art of treating such disordersin humans will be guided, from the data obtained in animal studies, tothe correct dosage and route of administration of the compound tohumans. In general, the determination of dosage and route ofadministration in humans is expected to be similar to that used todetermine administration in animals.

The identification of those patients who are in need of prophylactictreatment for disorders marked by degeneration of dopaminergic and/orGABAergic neurons is well within the ability and knowledge of oneskilled in the art. Certain of the methods for identification ofpatients which are at risk and which can be treated by the subjectmethod are appreciated in the medical arts, such as family history ofthe development of a particular disease state and the presence of riskfactors associated with the development of that disease state in thesubject patient. A clinician skilled in the art can readily identifysuch candidate patients, by the use of, for example, clinical tests,physical examination and medical/family history.

IV. Exemplary Hedgehog Therapeutic Compounds

The hedgehog therapeutic compositions of the subject method can begenerated by any of a variety of techniques, including purification ofnaturally occurring proteins, recombinantly produced proteins andsynthetic chemistry. Polypeptide forms of the hedgehog therapeutics arepreferably derived from vertebrate hedgehog proteins, e.g., havesequences corresponding to naturally occurring hedgehog proteins, orfragments thereof, from vertebrate organisms. However, it will beappreciated that the hedgehog polypeptide can correspond to a hedgehogprotein (or fragment thereof) which occurs in any metazoan organism.

The various naturally occurring hedgehog proteins from which the subjecttherapeutics can be derived are characterized by a signal peptide, ahighly conserved N-terminal region, and a more divergent C-terminaldomain. In addition to signal sequence cleavage in the secretory pathway(Lee, J. J. et al. (1992) Cell 71:33–50; Tabata, T. et al. (1992) GenesDev. 2635–2645; Chang, D. E. et al. (1994) Development 120:3339–3353),hedgehog precursor proteins naturally undergo an internalautoproteolytic cleavage which depends on conserved sequences in theC-terminal portion (Lee et al. (1994) Science 266:1528–1537; Porter etal. (1995) Nature 374:363–366). This autocleavage leads to a 19 kDN-terminal peptide and a C-terminal peptide of 26–28 kD (Lee et al.(1992) supra; Tabata et al. (1992) supra; Chang et al. (1994) supra; Leeet al. (1994) supra; Bumcrot, D. A., et al. (1995) Mol. Cell. Biol.15:2294–2303; Porter et al. (1995) supra; Ekker, S. C. et al. (1995)Curr. Biol. 5:944–955; Lai, C. J. et al. (1995) Development121:2349–2360). The N-terminal peptide stays tightly associated with thesurface of cells in which it was synthesized, while the C-terminalpeptide is freely diffusible both in vitro and in vivo (Lee et al.(1994) supra; Bumcrot et al. (1995) supra; Mart', E. et al. (1995)Development 121:2537–2547; Roelink, H. et al. (1995) Cell 81:445–455).Cell surface retention of the N-terminal peptide is dependent onautocleavage, as a truncated form of hedgehog encoded by an RNA whichterminates precisely at the normal position of internal cleavage isdiffusible in vitro (Porter et al. (1995) supra) and in vivo (Porter, J.A. et al. (1996) Cell 86, 21–34). Biochemical studies have shown thatthe autoproteolytic cleavage of the hedgehog precursor protein proceedsthrough an internal thioester intermediate which subsequently is cleavedin a nucleophilic substitution. It is suggested that the nucleophile isa small lipophilic molecule, more particularly cholesterol, whichbecomes covalently bound to the C-terminal end of the N-peptide (Porteret al. (1996) supra), tethering it to the cell surface.

The vertebrate family of hedgehog genes includes at least four members,e.g., paralogs of the single drosophila hedgehog gene (SEQ ID No. 19).Three of these members, herein referred to as Desert hedgehog (Dhh),Sonic hedgehog (Shh) and Indian hedgehog (Ihh), apparently exist in allvertebrates, including fish, birds, and mammals. A fourth member, hereinreferred to as tiggie-winkle hedgehog (Thh), appears specific to fish.According to the appended sequence listing, (see also Table 1) a chickenShh polypeptide is encoded by SEQ ID No:1; a mouse Dhh polypeptide isencoded by SEQ ID No:2; a mouse Ihh polypeptide is encoded by SEQ IDNo:3; a mouse Shh polypeptide is encoded by SEQ ID No:4 a zebrafish Shhpolypeptide is encoded by SEQ ID No:5; a human Shh polypeptide isencoded by SEQ ID No:6; a human Ihh polypeptide is encoded by SEQ IDNo:7; a human Dhh polypeptide is encoded by SEQ ID No. 8; and azebrafish Thh is encoded by SEQ ID No. 9.

TABLE 1 Guide to hedgehog sequences in Sequence Listing Nucleotide AminoAcid Chicken Shh SEQ ID No. 1 SEQ ID No. 10 Mouse Dhh SEQ ID No. 2 SEQID No. 11 Mouse Ihh SEQ ID No. 3 SEQ ID No. 12 Mouse Shh SEQ ID No. 4SEQ ID No. 13 Zebrafish Shh SEQ ID No. 5 SEQ ID No. 14 Human Shh SEQ IDNo. 6 SEQ ID No. 15 Human Ihh SEQ ID No. 7 SEQ ID No. 16 Human Dhh SEQID No. 8 SEQ ID No. 17 Zebrafish Thh SEQ ID No. 9 SEQ ID No. 18Drosophila HH SEQ ID No. 19 SEQ ID No. 20

In addition to the sequence variation between the various hedgehoghomologs, the hedgehog proteins are apparently present naturally in anumber of different forms, including a pro-form, a full-length matureform, and several processed fragments thereof. The pro-form includes anN-terminal signal peptide for directed secretion of the extracellulardomain, while the full-length mature form lacks this signal sequence.

As described above, further processing of the mature form occurs in someinstances to yield biologically active fragments of the protein. Forinstance, sonic hedgehog undergoes additional proteolytic processing toyield two peptides of approximately 19 kDa and 27 kDa, the 19 kDafragment corresponding to an proteolytic N-terminal portion of themature protein. In addition to proteolytic fragmentation and theaddition of one or more lipophilic groups according to the presentinvention, the hedgehog proteins can also be modifiedpost-translationally, such as by glycosylation and/or addition ofcholesterol, though bacterially produced (e.g.unglycosylated/uncholesterolized) forms of the proteins still maintaincertain of the bioactivities of the native protein. Bioactive fragmentsof hedgehog polypeptides of the present invention have been generatedand are described in great detail in, e.g., PCT publications WO 95/18856and WO 96/17924.

There are a wide range of lipophilic moieties with which hedgehogpolypeptides can be derivatized. The term “lipophilic group”, in thecontext of being attached to a hedgehog polypeptide, refers to a grouphaving high hydrocarbon content thereby giving the group high affinityto lipid phases. A lipophilic group can be, for example, a relativelylong chain alkyl or cycloalkyl (preferably n-alkyl) group havingapproximately 7 to 30 carbons. The alkyl group may terminate with ahydroxy or primary amine “tail”. To further illustrate, lipophilicmolecules include naturally-occurring and synthetic aromatic andnon-aromatic moieties such as fatty acids, esters and alcohols, otherlipid molecules, cage structures such as adamantane andbuckminsterfullerenes, and aromatic hydrocarbons such as benzene,perylene, phenanthrene, anthracene, naphthalene, pyrene, chrysene, andnaphthacene.

Particularly useful as lipophilic molecules are alicyclic hydrocarbons,saturated and unsaturated fatty acids and other lipid and phospholipidmoieties, waxes, cholesterol, isoprenoids, terpenes and polyalicyclichydrocarbons including adamantane and fullerenes, vitamins, polyethyleneglycol or oligoethylene glycol, (C₁–C₁₈)-alkyl phosphate diesters,—O—CH2-CH(OH)—O—(C12–C18)-alkyl, and in particular conjugates withpyrene derivatives. The lipophilic moiety can be a lipophilic dyesuitable for use in the invention include, but are not limited to,diphenylhexatriene, Nile Red, N-phenyl-1-naphthylamine, Prodan,Laurodan, Pyrene, Perylene, rhodamine, rhodamine B,tetramethylrhodamine, Texas Red, sulforhodamine,1,1′-didodecyl-3,3,3′,3′tetramethylindocarbocyanine perchlorate,octadecyl rhodamine B and the BODIPY dyes available from MolecularProbes Inc.

Other exemplary lipophilic moietites include aliphatic carbonyl radicalgroups include 1- or 2-adamantylacetyl, 3-methyladamant-1-ylacetyl,3-methyl-3-bromo-1-adamantylacetyl, 1-decalinacetyl, camphoracetyl,camphaneacetyl, noradamantylacetyl, norbornaneacetyl,bicyclo[2.2.2.]-oct-5-eneacetyl,1-methoxybicyclo[2.2.2.]-oct-5-ene-2-carbonyl,cis-5-norbornene-endo-2,3-dicarbonyl, 5-norbornen-2-ylacetyl,(1R)-myrtentaneacetyl, 2-norbornaneacetyl,anti-3-oxo-tricyclo[2.2.1.0<2,6>]-heptane-7-carbonyl, decanoyl,dodecanoyl, dodecenoyl, tetradecadienoyl, decynoyl or dodecynoyl.

Methods of Derivatizing the Hedgehog Polypeptide

The hedgehog polypeptide can be linked to the hydrophobic moiety in anumber of ways including by chemical coupling means, or by geneticengineering.

(i) Chemical Coupling Agents

There are a large number of chemical cross-linking agents that are knownto those skilled in the art. For the present invention, the preferredcross-linking agents are heterobifunctional cross-linkers, which can beused to link the hedgehog polypeptide and hydrophobic moiety in astepwise manner. Heterobifunctional cross-linkers provide the ability todesign more specific coupling methods for conjugating to proteins,thereby reducing the occurrences of unwanted side reactions such ashomo-protein polymers. A wide variety of heterobifunctionalcross-linkers are known in the art. These include:succinimidyl-4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (SMCC),m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS); N-succinimidyl(4-iodoacetyl) aminobenzoate (SIAB), succinimidyl-4-(p-maleimidophenyl)butyrate (SMPB), 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimidehydrochloride (EDC);4-succinimidyloxycarbonyl-α-methyl-α-(2-pyridyldithio)-toluene (SMPT),N-succinimidyl-3-(2-pyridyldithio) propionate (SPDP), succinimidyl6-[3-(2-pyridyldithio)-propionate]hexanoate (LC-SPDP). Thosecross-linking agents having N-hydroxysuccinimide moieties can beobtained as the N-hydroxysulfosuccinimide analogs, which generally havegreater water solubility. In addition, those cross-linking agents havingdisulfide bridges within the linking chain can be synthesized instead asthe alkyl derivatives so as to reduce the amount of linker cleavage invivo.

In addition to the heterobifunctional cross-linkers, there exists anumber of other cross-linking agents including homobifunctional andphotoreactive cross-linkers. Disuccinimidyl suberate (DSS),bismaleimidohexane (BMH) and dimethylpimelimidate-2 HCl (DMP) areexamples of useful homobifunctional cross-linking agents, andbis-[B-(4-azidosalicylamido)ethyl]disulfide (BASED) andN-succinimidyl-6(4′-azido-2′-nitrophenylamino)hexanoate (SANPAH) areexamples of useful photoreactive cross-linkers for use in thisinvention. For a recent review of protein coupling techniques, see Meanset al. (1990) Bioconjugate Chemistry 1:2–12, incorporated by referenceherein.

One particularly useful class of heterobifunctional cross-linkers,included above, contain the primary amine reactive group,N-hydroxysuccinimide (NHS), or its water soluble analogN-hydroxysulfosuccinimide (sulfo-NHS). Primary amines (lysine epsilongroups) at alkaline pH's are unprotonated and react by nucleophilicattack on NHS or sulfo-NHS esters. This reaction results in theformation of an amide bond, and release of NHS or sulfo-NHS as aby-product.

Another reactive group useful as part of a heterobifunctionalcross-linker is a thiol reactive group. Common thiol reactive groupsinclude maleimides, halogens, and pyridyl disulfides. Maleimides reactspecifically with free sulfhydryls (cysteine residues) in minutes, underslightly acidic to neutral (pH 6.5–7.5) conditions. Halogens (iodoacetylfunctions) react with —SH groups at physiological pH's. Both of thesereactive groups result in the formation of stable thioether bonds.

The third component of the heterobifunctional cross-linker is the spacerarm or bridge. The bridge is the structure that connects the tworeactive ends. The most apparent attribute of the bridge is its effecton steric hindrance. In some instances, a longer bridge can more easilyspan the distance necessary to link two complex biomolecules. Forinstance, SMPB has a span of 14.5 angstroms.

Preparing protein-protein conjugates using heterobifunctional reagentsis a two-step process involving the amine reaction and the sulfhydrylreaction. For the first step, the amine reaction, the protein chosenshould contain a primary amine. This can be lysine epsilon amines or aprimary alpha amine found at the N-terminus of most proteins. Theprotein should not contain free sulfhydryl groups. In cases where bothproteins to be conjugated contain free sulfhydryl groups, one proteincan be modified so that all sulfhydryls are blocked using for instance,N-ethylmaleimide (see Partis et al. (1983) J. Pro. Chem. 2:263,incorporated by reference herein). Ellman's Reagent can be used tocalculate the quantity of sulfhydryls in a particular protein (see forexample Ellman et al. (1958) Arch. Biochem. Biophys. 74:443 and Riddleset al. (1979) Anal. Biochem. 94:75, incorporated by reference herein).

The reaction buffer should be free of extraneous amines and sulfhydryls.The pH of the reaction buffer should be 7.0–7.5. This pH range preventsmaleimide groups from reacting with amines, preserving the maleimidegroup for the second reaction with sulfhydryls.

The NHS-ester containing cross-linkers have limited water solubility.They should be dissolved in a minimal amount of organic solvent (DMF orDMSO) before introducing the cross-linker into the reaction mixture. Thecross-linker/solvent forms an emulsion which will allow the reaction tooccur.

The sulfo-NHS ester analogs are more water soluble, and can be addeddirectly to the reaction buffer. Buffers of high ionic strength shouldbe avoided, as they have a tendency to “salt out” the sulfo-NHS esters.To avoid loss of reactivity due to hydrolysis, the cross-linker is addedto the reaction mixture immediately after dissolving the proteinsolution.

The reactions can be more efficient in concentrated protein solutions.The more alkaline the pH of the reaction mixture, the faster the rate ofreaction. The rate of hydrolysis of the NHS and sulfo-NHS esters willalso increase with increasing pH. Higher temperatures will increase thereaction rates for both hydrolysis and acylation.

Once the reaction is completed, the first protein is now activated, witha sulfhydryl reactive moiety. The activated protein may be isolated fromthe reaction mixture by simple gel filtration or dialysis. To carry outthe second step of the cross-linking, the sulfhydryl reaction, thelipophilic group chosen for reaction with maleimides, activatedhalogens, or pyridyl disulfides must contain a free sulfhydryl.Alternatively, a primary amine may be modified with to add a sulfhydryl

In all cases, the buffer should be degassed to prevent oxidation ofsulfhydryl groups. EDTA may be added to chelate any oxidizing metalsthat may be present in the buffer. Buffers should be free of anysulfhydryl containing compounds.

Maleimides react specifically with —SH groups at slightly acidic toneutral pH ranges (6.5–7.5). A neutral pH is sufficient for reactionsinvolving halogens and pyridyl disulfides. Under these conditions,maleimides generally react with —SH groups within a matter of minutes.Longer reaction times are required for halogens and pyridyl disulfides.

The first sulfhydryl reactive-protein prepared in the amine reactionstep is mixed with the sulfhydryl-containing lipophilic group under theappropriate buffer conditions. The conjugates can be isolated from thereaction mixture by methods such as gel filtration or by dialysis.

Exemplary activated lipophilic moieties for conjugation include:N-(1-pyrene)maleimide; 2,5-dimethoxystilbene-4′-maleimide,eosin-5-maleimide; fluorescein-5-maleimide;N-(4-(6-dimethylamino-2-benzofuranyl)phenyl)maleimide;benzophenone-4-maleimide; 4-dimethylaminophenylazophenyl-4′-maleimide(DABMI), tetramethylrhodamine-5-maleimide,tetramethylrhodamine-6-maleimide, Rhodamine Red™ C2 maleimide,N-(5-aminopentyl)maleimide, trifluoroacetic acid salt,N-(2-aminoethyl)maleimide, trifluoroacetic acid salt, Oregon Green™ 488maleimide,N-(2-((2-(((4-azido-2,3,5,6-tetrafluoro)benzoyl)amino)ethyl)dithio)ethyl)maleimide(TFPAM-SS1),2-(1-(3-dimethylaminopropyl)-indol-3-yl)-3-(indol-3-yl)maleimide(bisindolylmaleimide; GF 109203X), BODIPY® FL N-(2-aminoethyl)maleimide,N-(7-dimethylamino-4-methylcoumarin-3-yl)maleimide (DACM), Alexa™ 488 C5maleimide, Alexa™ 594 C5 maleimide, sodium saltN-(1-pyrene)maleimide,2,5-dimethoxystilbene-4′-maleimide, eosin-5-maleimide,fluorescein-5-maleimide,N-(4-(6-dimethylamino-2-benzofuranyl)phenyl)maleimide,benzophenone-4-maleimide, 4-dimethylaminophenylazophenyl-4′-maleimide,1-(2-maleimidylethyl)-4-(5-(4-methoxyphenyl)oxazol-2-yl)pyridiniummethanesulfonate, tetramethylrhodamine-5-maleimide,tetramethylrhodamine-6-maleimide, Rhodamine Red™ C2 maleimide,N-(5-aminopentyl)maleimide, N-(2-aminoethyl)maleimide,N-(2-((2-(((4-azido-2,3,5,6-tetrafluoro)benzoyl)-amino)ethyl)dithio)ethyl)maleimide,2-(1-(3-dimethylaminopropyl)-indol-3-yl)-3-(indol-3-yl) maleimide,N-(7-dimethylamino-4-methylcoumarin-3-yl)maleimide (DACM),11H-Benzo[α]fluorene, Benzo[α]pyrene.

In one embodiment, the hedgehog polypeptide can be derivatived usingpyrene maleimide, which can be purchased from Molecular Probes (Eugene,Oreg.), e.g., N-(1-pyrene)maleimide or 1-pyrenemethyl iodoacetate (PMIAester). As illustrated in FIG. 1, the pyrene-derived hedgehog proteinhad an activity profile indicating that it was nearly 2 orders ofmagnitude more active than the unmodified form of the protein.

For those embodiments wherein the hydophobic moiety is a polypeptide,the modified hedgehog polypeptide of this invention can be constructedas a fusion protein, containing the hedgehog polypeptide and thehydrophobic moiety as one contiguous polypeptide chain.

In certain embodiments, the lipophilic moiety is an amphipathicpolypeptide, such as magainin, cecropin, attacin, melittin, gramicidinS, alpha-toxin of Staph. aureus, alamethicin or a synthetic amphipathicpolypeptide. Fusogenic coat proteins from viral particles can also be aconvenient source of amphipathic sequences for the subject hedgehogproteins.

The present application is directed to the discovery that, in additionto those effects seen by cholesterol-addition to the C-terminus ofextracellular fragments of the protein, at least certain of thebiological activities of the hedgehog gene products are unexpectedlypotentiated by derivativation of the protein with lipophilic moieties atother sites on the protein and/or by moieties other than cholesterol.Certain aspects of the invention are directed to preparations ofhedgehog polypeptides which are modified at sites other than N-terminalor C-terminal residues of the natural processed form of the protein,and/or which are modified at such terminal residues with lipophilicmoieties other than a sterol at the C-terminus or fatty acid at theN-terminus.

Moreover, mutagenesis can be used to create modified hh polypeptides,e.g., for such purposes as enhancing therapeutic or prophylacticefficacy, or stability (e.g., ex vivo shelf life and resistance toproteolytic degradation in vivo). Such modified peptides can beproduced, for instance, by amino acid substitution, deletion, oraddition. Modified hedgehog polypeptides can also include those withaltered post-translational processing relative to a naturally occurringhedgehog protein, e.g., altered glycosylation, cholesterolization,prenylation and the like.

In one embodiment, the hedgehog therapeutic is a polypeptide encodableby a nucleotide sequence that hybridizes under stringent conditions to ahedgehog coding sequence represented in one or more of SEQ ID Nos:1–9 or19. Appropriate stringency conditions which promote DNA hybridization,for example, 6.0× sodium chloride/sodium citrate (SSC) at about 45° C.,followed by a wash of 2.0×SSC at 50° C., are known to those skilled inthe art or can be found in Current Protocols in Molecular Biology, JohnWiley & Sons, N.Y. (1989), 6.3.1–6.3.6. For example, the saltconcentration in the wash step can be selected from a low stringency ofabout 2.0×SSC at 50° C. to a high stringency of about 0.2×SSC at 50° C.In addition, the temperature in the wash step can be increased from lowstringency conditions at room temperature, about 22° C., to highstringency conditions at about 65° C.

As described in the literature, genes for other hedgehog proteins, e.g.,from other animals, can be obtained from mRNA or genomic DNA samplesusing techniques well known in the art. For example, a cDNA encoding ahedgehog protein can be obtained by isolating total mRNA from a cell,e.g. a mammalian cell, e.g. a human cell, including embryonic cells.Double stranded cDNAs can then be prepared from the total mRNA, andsubsequently inserted into a suitable plasmid or bacteriophage vectorusing any one of a number of known techniques. The gene encoding ahedgehog protein can also be cloned using established polymerase chainreaction techniques.

Preferred nucleic acids encode a hedgehog polypeptide comprising anamino acid sequence at least 60% homologous, more preferably 70%homologous and most preferably 80% homologous with an amino acidsequence selected from the group consisting of SEQ ID Nos:8–14. Nucleicacids which encode polypeptides at least about 90%, more preferably atleast about 95%, and most preferably at least about 98–99% homology withan amino acid sequence represented in one of SEQ ID Nos:10–18 or 20 arealso within the scope of the invention.

Hedgehog polypeptides preferred by the present invention, in addition tonative hedgehog proteins, are at least 60% homologous, more preferably70% homologous and most preferably 80% homologous with an amino acidsequence represented by any of SEQ ID Nos:10–18 or 20. Polypeptideswhich are at least 90%, more preferably at least 95%, and mostpreferably at least about 98–99% homologous with a sequence selectedfrom the group consisting of SEQ ID Nos:10–18 or 20 are also within thescope of the invention. The only prerequisite is that the hedgehogpolypeptide is capable of protecting neuronal cells againstdegeneration, e.g., the polypeptide is trophic for a dopaminergic and/orGABAergic neuron.

The term “recombinant protein” refers to a polypeptide of the presentinvention which is produced by recombinant DNA techniques, whereingenerally, DNA encoding a hedgehog polypeptide is inserted into asuitable expression vector which is in turn used to transform a hostcell to produce the heterologous protein. Moreover, the phrase “derivedfrom”, with respect to a recombinant hedgehog gene, is meant to includewithin the meaning of “recombinant protein” those proteins having anamino acid sequence of a native hedgehog protein, or an amino acidsequence similar thereto which is generated by mutations includingsubstitutions and deletions (including truncation) of a naturallyoccurring form of the protein.

The method of the present invention can also be carried out usingvariant forms of the naturally occurring hedgehog polypeptides, e.g.,mutational variants.

As is known in the art, hedgehog polypeptides can be produced bystandard biological techniques. For example, a host cell transfectedwith a nucleic acid vector directing expression of a nucleotide sequenceencoding the subject polypeptides can be cultured under appropriateconditions to allow expression of the peptide to occur. The polypeptidehedgehog may be secreted and isolated from a mixture of cells and mediumcontaining the recombinant hedgehog polypeptide. Alternatively, thepeptide may be retained cytoplasmically by removing the signal peptidesequence from the recombinant hedgehog gene and the cells harvested,lysed and the protein isolated. A cell culture includes host cells,media and other byproducts. Suitable media for cell culture are wellknown in the art. The recombinant hedgehog polypeptide can be isolatedfrom cell culture medium, host cells, or both using techniques known inthe art for purifying proteins including ion-exchange chromatography,gel filtration chromatography, ultrafiltration, electrophoresis, andimmunoaffinity purification with antibodies specific for such peptide.In a preferred embodiment, the recombinant hedgehog polypeptide is afusion protein containing a domain which facilitates its purification,such as an hedgehog/GST fusion protein. The host cell may be anyprokaryotic or eukaryotic cell.

Recombinant hedgehog genes can be produced by ligating nucleic acidencoding an hedgehog protein, or a portion thereof, into a vectorsuitable for expression in either prokaryotic cells, eukaryotic cells,or both. Expression vectors for production of recombinant forms of thesubject hedgehog polypeptides include plasmids and other vectors. Forinstance, suitable vectors for the expression of a hedgehog polypeptideinclude plasmids of the types: pBR322-derived plasmids, pEMBL-derivedplasmids, pEX-derived plasmids, pBTac-derived plasmids and pUC-derivedplasmids for expression in prokaryotic cells, such as E. coli.

A number of vectors exist for the expression of recombinant proteins inyeast. For instance, YEP24, YIP5, YEP51, YEP52, pYES2, and YRP17 arecloning and expression vehicles useful in the introduction of geneticconstructs into S. cerevisiae (see, for example, Broach et al. (1983) inExperimental Manipulation of Gene Expression, ed. M. Inouye AcademicPress, p. 83, incorporated by reference herein). These vectors canreplicate in E. coli due the presence of the pBR322 ori, and in S.cerevisiae due to the replication determinant of the yeast 2 micronplasmid. In addition, drug resistance markers such as ampicillin can beused. In an illustrative embodiment, an hedgehog polypeptide is producedrecombinantly utilizing an expression vector generated by sub-cloningthe coding sequence of one of the hedgehog genes represented in SEQ IDNos:1–9 or 19.

The preferred mammalian expression vectors contain both prokaryoticsequences, to facilitate the propagation of the vector in bacteria, andone or more eukaryotic transcription units that are expressed ineukaryotic cells. The pcDNAI/amp, pcDNAI/neo, pRc/CMV, pSV2gpt, pSV2neo,pSV2-dhfr, pTk2, pRSVneo, pMSG, pSVT7, pko-neo and pHyg derived vectorsare examples of mammalian expression vectors suitable for transfectionof eukaryotic cells. Some of these vectors are modified with sequencesfrom bacterial plasmids, such as pBR322, to facilitate replication anddrug resistance selection in both prokaryotic and eukaryotic cells.Alternatively, derivatives of viruses such as the bovine papillomavirus(BPV-1), or Epstein-Barr virus (pHEBo, pREP-derived and p205) can beused for transient expression of proteins in eukaryotic cells. Thevarious methods employed in the preparation of the plasmids andtransformation of host organisms are well known in the art. For othersuitable expression systems for both prokaryotic and eukaryotic cells,as well as general recombinant procedures, see Molecular Cloning ALaboratory Manual, 2nd Ed., ed. by Sambrook, Fritsch and Maniatis (ColdSpring Harbor Laboratory Press: 1989) Chapters 16 and 17.

In some instances, it may be desirable to express the recombinanthedgehog polypeptide by the use of a baculovirus expression system.Examples of such baculovirus expression systems include pVL-derivedvectors (such as pVL1392, pVL1393 and pVL941), pAcUW-derived vectors(such as pAcUW1), and pBlueBac-derived vectors (such as the β-galcontaining pBlueBac III).

When it is desirable to express only a portion of a hedgehog protein,such as a form lacking a portion of the N-terminus, i.e., a truncationmutant which lacks the signal peptide, it may be necessary to add astart codon (ATG) to the oligonucleotide fragment containing the desiredsequence to be expressed. It is well known in the art that a methionineat the N-terminal position can be enzymatically cleaved by the use ofthe enzyme methionine aminopeptidase (MAP). MAP has been cloned from E.coli (Ben-Bassat et al. (1987) J. Bacteriol. 169:751–757) and Salmonellatyphimurium and its in vitro activity has been demonstrated onrecombinant proteins (Miller et al. (1987) PNAS 84:2718–1722).Therefore, removal of an N-terminal methionine, if desired, can beachieved either in vivo by expressing hedgehog-derived polypeptides in ahost which produces MAP (e.g., E. coli or CM89 or S. cerevisiae), or invitro by use of purified MAP (e.g., procedure of Miller et al., supra).

Alternatively, the coding sequences for the polypeptide can beincorporated as a part of a fusion gene including a nucleotide sequenceencoding a different polypeptide. It is widely appreciated that fusionproteins can also facilitate the expression of proteins, andaccordingly, can be used in the expression of the hedgehog polypeptidesof the present invention. For example, hedgehog polypeptides can begenerated as glutathione-S-transferase (GST-fusion) proteins. SuchGST-fusion proteins can enable easy purification of the hedgehogpolypeptide, as for example by the use of glutathione-derivatizedmatrices (see, for example, Current Protocols in Molecular Biology, eds.Ausubel et al. (N.Y.: John Wiley & Sons, 1991)). In another embodiment,a fusion gene coding for a purification leader sequence, such as apoly-(His)/enterokinase cleavage site sequence, can be used to replacethe signal sequence which naturally occurs at the N-terminus of thehedgehog protein (e.g., of the pro-form, in order to permit purificationof the poly(His)-hedgehog protein by affinity chromatography using aNi²⁺ metal resin. The purification leader sequence can then besubsequently removed by treatment with enterokinase (e.g., see Hochuliet al. (1987) J. Chromatography 411:177; and Janknecht et al. PNAS88:8972).

Techniques for making fusion genes are known to those skilled in theart. Essentially, the joining of various DNA fragments coding fordifferent polypeptide sequences is performed in accordance withconventional techniques, employing blunt-ended or stagger-ended terminifor ligation, restriction enzyme digestion to provide for appropriatetermini, filling-in of cohesive ends as appropriate, alkalinephosphatase treatment to avoid undesirable joining, and enzymaticligation. In another embodiment, the fusion gene can be synthesized byconventional techniques including automated DNA synthesizers.Alternatively, PCR amplification of gene fragments can be carried outusing anchor primers which give rise to complementary overhangs betweentwo consecutive gene fragments which can subsequently be annealed togenerate a chimeric gene sequence (see, for example, Current Protocolsin Molecular Biology, eds. Ausubel et al. John Wiley & Sons: 1992).

Hedgehog polypeptides may also be chemically modified to create hedgehogderivatives by forming covalent or aggregate conjugates with otherchemical moieties, such as glycosyl groups, cholesterol, isoprenoids,lipids, phosphate, acetyl groups and the like. Covalent derivatives ofhedgehog proteins can be prepared by linking the chemical moieties tofunctional groups on amino acid sidechains of the protein or at theN-terminus or at the C-terminus of the polypeptide.

For instance, hedgehog proteins can be generated to include a moiety,other than sequence naturally associated with the protein, that binds acomponent of the extracellular matrix and enhances localization of theanalog to cell surfaces. For example, sequences derived from thefibronectin “type-III repeat”, such as a tetrapeptide sequence R-G-D-S(Pierschbacher et al. (1984) Nature 309:30–3; and Kornblihtt et al.(1985) EMBO 4:1755–9) can be added to the hedgehog polypeptide tosupport attachment of the chimeric molecule to a cell through bindingECM components (Ruoslahti et al. (1987) Science 238:491–497;Pierschbacheret al. (1987) J. Biol. Chem. 262:17294–8.; Hynes (1987)Cell 48:549–54; and Hynes (1992) Cell 69:11–25).

In preferred embodiment, the hedgehog polypeptide is isolated from, oris otherwise substantially free of, other cellular proteins, especiallyother extracellular or cell surface associated proteins which maynormally be associated with the hedgehog polypeptide. The term“substantially free of other cellular or extracellular proteins” (alsoreferred to herein as “contaminating proteins”) or “substantially pureor purified preparations” are defined as encompassing preparations ofhedgehog polypeptides having less than 20% (by dry weight) contaminatingprotein, and preferably having less than 5% contaminating protein. By“purified”, it is meant that the indicated molecule is present in thesubstantial absence of other biological macromolecules, such as otherproteins. The term “purified” as used herein preferably means at least80% by dry weight, more preferably in the range of 95–99% by weight, andmost preferably at least 99.8% by weight, of biological macromoleculesof the same type present (but water, buffers, and other small molecules,especially molecules having a molecular weight of less than 5000, can bepresent). The term “pure” as used herein preferably has the samenumerical limits as “purified” immediately above.

As described above for recombinant polypeptides, isolated hedgehogpolypeptides can include all or a portion of the amino acid sequencesrepresented in any of SEQ ID Nos:10–18 or 20, or a homologous sequencethereto. Preferred fragments of the subject hedgehog proteins correspondto the N-terminal and C-terminal proteolytic fragments of the matureprotein. Bioactive fragments of hedgehog polypeptides are described ingreat detail in PCT publications WO 95/18856 and WO 96/17924.

With respect to bioctive fragments of hedgehog polypeptide, preferredhedgehog therapeutics include at least 50 amino acid residues of ahedgehog polypeptide, more preferably at least 100, and even morepreferably at least 150.

Another preferred hedgehog polypeptide which can be included in thehedgehog therapeutic is an N-terminal fragment of the mature proteinhaving a molecular weight of approximately 19 kDa.

Preferred human hedgehog proteins include N-terminal fragmentscorresponding approximately to residues 24–197 of SEQ ID No. 15, 28–202of SEQ ID No. 16, and 23–198 of SEQ ID No. 17. By “correspondingapproximately” it is meant that the sequence of interest is at most 20amino acid residues different in length to the reference sequence,though more preferably at most 5, 10 or 15 amino acid different inlength.

Still other preferred hedgehog polypeptides include an amino acidsequence represented by the formula A-B wherein: (i) A represents all orthe portion of the amino acid sequence designated by residues 1–168 ofSEQ ID No:21; and B represents at least one amino acid residue of theamino acid sequence designated by residues 169–221 of SEQ ID No:21; (ii)A represents all or the portion of the amino acid sequence designated byresidues 24–193 of SEQ ID No:15; and B represents at least one aminoacid residue of the amino acid sequence designated by residues 194–250of SEQ ID No:15; (iii) A represents all or the portion of the amino acidsequence designated by residues 25–193 of SEQ ID No:13; and B representsat least one amino acid residue of the amino acid sequence designated byresidues 194–250 of SEQ ID No:13; (iv) A represents all or the portionof the amino acid sequence designated by residues 23–193 of SEQ IDNo:11; and B represents at least one amino acid residue of the aminoacid sequence designated by residues 194–250 of SEQ ID No:11; (v) Arepresents all or the portion of the amino acid sequence designated byresidues 28–197 of SEQ ID No:12; and B represents at least one aminoacid residue of the amino acid sequence designated by residues 198–250of SEQ ID No:12; (vi) A represents all or the portion of the amino acidsequence designated by residues 29–197 of SEQ ID No:16; and B representsat least one amino acid residue of the amino acid sequence designated byresidues 198–250 of SEQ ID No:16; or (vii) A represents all or theportion of the amino acid sequence designated by residues 23–193 of SEQID No. 17, and B represents at least one amino acid residue of the aminoacid sequence designated by residues 194–250 of SEQ ID No. 17. Incertain preferred embodiments, A and B together represent a contiguouspolypeptide sequence of the designated sequence, A represents at least25, 50, 75, 100, 125 or 150 amino acids of the designated sequence, andB represents at least 5, 10, or 20 amino acid residues of the amino acidsequence designated by corresponding entry in the sequence listing, andA and B together preferably represent a contiguous sequencecorresponding to the sequence listing entry. Similar fragments fromother hedgehog proteins are also contemplated, e.g., fragments whichcorrespond to the preferred fragments from the sequence listing entrieswhich are enumerated above.

Isolated peptidyl portions of hedgehog proteins can be obtained byscreening peptides recombinantly produced from the correspondingfragment of the nucleic acid encoding such peptides. In addition,fragments can be chemically synthesized using techniques known in theart such as conventional Merrifield solid phase f-Moc or t-Bocchemistry. For example, a hedgehog polypeptide of the present inventionmay be arbitrarily divided into fragments of desired length with nooverlap of the fragments, or preferably divided into overlappingfragments of a desired length. The fragments can be produced(recombinantly or by chemical synthesis) and tested to identify thosepeptidyl fragments which can function as agonists of a wild-type (e.g.,“authentic”) hedgehog protein. For example, Roman et al. (1994) Eur JBiochem 222:65–73 describe the use of competitive-binding assays usingshort, overlapping synthetic peptides from larger proteins to identifybinding domains.

The recombinant hedgehog polypeptides of the present invention alsoinclude homologs of the authentic hedgehog proteins, such as versions ofthose protein which are resistant to proteolytic cleavage, as forexample, due to mutations which alter potential cleavage sequences orwhich inactivate an enzymatic activity associated with the protein.Hedgehog homologs of the present invention also include proteins whichhave been post-translationally modified in a manner different than theauthentic protein. Exemplary derivatives of hedgehog proteins includepolypeptides which lack glycosylation sites (e.g. to produce anunglycosylated protein), which lack sites for cholesterolization, and/orwhich lack N-terminal and/or C-terminal sequences.

Modification of the structure of the subject hedgehog polypeptides canalso be for such purposes as enhancing therapeutic or prophylacticefficacy, or stability (e.g., ex vivo shelf life and resistance toproteolytic degradation in vivo). Such modified peptides, when designedto retain at least one activity of the naturally-occurring form of theprotein, are considered functional equivalents of the hedgehogpolypeptides described in more detail herein. Such modified peptides canbe produced, for instance, by amino acid substitution, deletion, oraddition.

It is well known in the art that certain isolated replacements of aminoacids, e.g., replacement of an amino acid residue with another relatedamino acid (i.e. isosteric and/or isoelectric mutations), can be carriedout without major effect on the biological activity of the resultingmolecule. Conservative replacements are those that take place within afamily of amino acids that are related in their side chains. Geneticallyencoded amino acids are can be divided into four families: (1)acidic=aspartate, glutamate; (2) basic=lysine, arginine, histidine; (3)nonpolar=alanine, valine, leucine, isoleucine, proline, phenylalanine,methionine, tryptophan; and (4) uncharged polar=glycine, asparagine,glutamine, cysteine, serine, threonine, tyrosine. Phenylalanine,tryptophan, and tyrosine are sometimes classified jointly as aromaticamino acids. In similar fashion, the amino acid repertoire can begrouped as (1) acidic=aspartate, glutamate; (2) basic=lysine, argininehistidine, (3) aliphatic=glycine, alanine, valine, leucine, isoleucine,serine, threonine, with serine and threonine optionally be groupedseparately as aliphatic-hydroxyl; (4) aromatic=phenylalanine, tyrosine,tryptophan; (5) amide=asparagine, glutamine; and (6)sulfur-containing=cysteine and methionine. (see, for example,Biochemistry, 2nd ed., Ed. by L. Stryer, WH Freeman and Co.: 1981).Whether a change in the amino acid sequence of a peptide results in afunctional hedgehog homolog (e.g. functional in the sense that it actsto mimic or antagonize the wild-type form) can be readily determined byassessing the ability of the variant peptide to produce a response incells in a fashion similar to the wild-type protein, or competitivelyinhibit such a response. Polypeptides in which more than one replacementhas taken place can readily be tested in the same manner.

It is specifically contemplated that the methods of the presentinvention can be carried using homologs of naturally occurring hedgehogproteins. In one embodiment, the invention contemplates using hedgehogpolypeptides generated by combinatorial mutagenesis. Such methods, asare known in the art, are convenient for generating both point andtruncation mutants, and can be especially useful for identifyingpotential variant sequences (e.g. homologs) that are functional inbinding to a receptor for hedgehog proteins. The purpose of screeningsuch combinatorial libraries is to generate, for example, novel hedgehoghomologs which can act as neuroprotective agents. To illustrate,hedgehog homologs can be engineered by the present method to providemore efficient binding to a cognate receptor, such as patched, retainingneuroprotective activity. Thus, combinatorially-derived homologs can begenerated to have an increased potency relative to a naturally occurringform of the protein. Moreover, manipulation of certain domains ofhedgehog by the present method can provide domains more suitable for usein fusion proteins, such as one that incorporates portions of otherproteins which are derived from the extracellular matrix and/or whichbind extracellular matrix components.

To further illustrate the state of the art of combinatorial mutagenesis,it is noted that the review article of Gallop et al. (1994) J Med Chem37:1233 describes the general state of the art of combinatoriallibraries as of the earlier 1990's. In particular, Gallop et al state atpage 1239 “[s]creening the analog libraries aids in determining theminimum size of the active sequence and in identifying those residuescritical for binding and intolerant of substitution”. In addition, theLadner et al. PCT publication WO90/02809, the Goeddel et al. U.S. Pat.No. 5,223,408, and the Markland et al. PCT publication WO92/15679illustrate specific techniques which one skilled in the art couldutilize to generate libraries of hedgehog variants which can be rapidlyscreened to identify variants/fragments which retained a particularactivity of the hedgehog polypeptides. These techniques are exemplary ofthe art and demonstrate that large libraries of relatedvariants/truncants can be generated and assayed to isolate particularvariants without undue experimentation. Gustin et al. (1993) Virology193:653, and Bass et al. (1990) Proteins: Structure, Function andGenetics 8:309–314 also describe other exemplary techniques from the artwhich can be adapted as means for generating mutagenic variants ofhedgehog polypeptides.

Indeed, it is plain from the combinatorial mutagenesis art that largescale mutagenesis of hedgehog proteins, without any preconceived ideasof which residues were critical to the biological function, and generatewide arrays of variants having equivalent biological activity. Indeed,it is the ability of combinatorial techniques to screen billions ofdifferent variants by high throughout analysis that removes anyrequirement of a priori understanding or knowledge of critical residues.

To illsutrate, the amino acid sequences for a population of hedgehoghomologs or other related proteins are aligned, preferably to promotethe highest homology possible. Such a population of variants caninclude, for example, hedgehog homologs from one or more species. Aminoacids which appear at each position of the aligned sequences areselected to create a degenerate set of combinatorial sequences. In apreferred embodiment, the variegated library of hedgehog variants isgenerated by combinatorial mutagenesis at the nucleic acid level, and isencoded by a variegated gene library. For instance, a mixture ofsynthetic oligonucleotides can be enzymatically ligated into genesequences such that the degenerate set of potential hedgehog sequencesare expressible as individual polypeptides, or alternatively, as a setof larger fusion proteins (e.g. for phage display) containing the set ofhedgehog sequences therein.

As illustrated in PCT publication WO 95/18856, to analyze the sequencesof a population of variants, the amino acid sequences of interest can bealigned relative to sequence homology. The presence or absence of aminoacids from an aligned sequence of a particular variant is relative to achosen consensus length of a reference sequence, which can be real orartificial.

In an illustrative embodiment, alignment of exons 1, 2 and a portion ofexon 3 encoded sequences (e.g. the N-terminal approximately 221 residuesof the mature protein) of each of the Shh clones produces a degenerateset of Shh polypeptides represented by the general formula:

(SEQ ID No:21) C-G-P-G-R-G-X(1)-G-X-(2)-R-R-H-P-K-K-L-T-P-L-A-Y-K-Q-F-I-P-N-V-A-E-K-T-L-G-A-S-G-R-Y-E-G-K-I-X(3)-R-N-S-E-R-F-K-E-L-T-P-N-Y-N-P-D-I-I-F-K-D-E-E-N-T-G-A-D-R-L-M-T-Q-R-C-K-D-K-L-M-T-Q-R-C-K-D-K-L-N-X(4)-L-A-I-S-V-M-N-X(5)-W-P-G-V-X(6)-L-R-V-T-E-G-W-D-E-D-G-H-H-X(7)-E-E-S-L-H-Y-E-G-R-A-V-D-I-T-T-S-D-R-D-X(8)-S-K-Y-G-X(9)-L-X(10)-R-L-A-V-E-A-G-F-D-W-V-Y-Y-E-S-K-A-H-I-H-C-S-V-K-A-E-N-S-V-A-A-K-S-G-G-C-F-P-G-S-A-X(11)-V-X(12)-L-X(13)-X(14)-G-G-X(15)-K-X-(16)-V-K-D-L-X(17)-P-G-D-X(18)-V-L-A-A-D-X(19)-X(20)-G-X(21)-L-X(22)-X(23)-S-D-F-X(24)-X (25)-F-X(26)-D-R,wherein each of the degenerate positions “X” can be an amino acid whichoccurs in that position in one of the human, mouse, chicken or zebrafishShh clones, or, to expand the library, each X can also be selected fromamongst amino acid residue which would be conservative substitutions forthe amino acids which appear naturally in each of those positions. Forinstance, Xaa(1) represents Gly, Ala, Val, Leu, Ile, Phe, Tyr or Trp;Xaa(2) represents Arg, His or Lys; Xaa(3) represents Gly, Ala, Val, Leu,Ile, Ser or Thr; Xaa(4) represents Gly, Ala, Val, Leu, Ile, Ser or Thr;Xaa(5) represents Lys, Arg, His, Asn or Gln; Xaa(6) represents Lys, Argor His; Xaa(7) represents Ser, Thr, Tyr, Trp or Phe; Xaa(8) representsLys, Arg or His; Xaa(9) represents Met, Cys, Ser or Thr; Xaa(10)represents Gly, Ala, Val, Leu, Ile, Ser or Thr; Xaa(11) represents Leu,Val, Met, Thr or Ser; Xaa(12) represents His, Phe, Tyr, Ser, Thr, Met orCys; Xaa(13) represents Gln, Asn, Glu, or Asp; Xaa(14) represents His,Phe, Tyr, Thr, Gln, Asn, Glu or Asp; Xaa(15) represents Gln, Asn, Glu,Asp, Thr, Ser, Met or Cys; Xaa(16) represents Ala, Gly, Cys, Leu, Val orMet; Xaa(17) represents Arg, Lys, Met, Ile, Asn, Asp, Glu, Gln, Ser, Thror Cys; Xaa(18) represents Arg, Lys, Met or Ile; Xaa(19) represents Ala,Gly, Cys, Asp, Glu, Gln, Asn, Ser, Thr or Met; Xaa(20) represents Ala,Gly, Cys, Asp, Asn, Glu or Gln; Xaa(21) represents Arg, Lys, Met, Ile,Asn, Asp, Glu or Gln; Xaa(22) represent Leu, Val, Met or Ile; Xaa(23)represents Phe, Tyr, Thr, His or Trp; Xaa(24) represents Ile, Val, Leuor Met; Xaa(25) represents Met, Cys, Ile, Leu, Val, Thr or Ser; Xaa(26)represents Leu, Val, Met, Thr or Ser. In an even more expansive library,each X can be selected from any amino acid.

In similar fashion, alignment of each of the human, mouse, chicken andzebrafish hedgehog clones, can provide a degenerate polypeptide sequencerepresented by the general formula:

(SEQ ID No:22) C-G-P-G-R-G-X(1)-X(2)-X(3)-R-R-X(4)-X(5)-X(6)-P-K-X(7)-L-X(8)-P-L-X(9)-Y-K-Q-F-X(10)-P-X(11)-X(12)-X(13)-E-X(14)-T-L-G-A-S-G-X(15)-X(16)-E-G-X(17)-X(18)-X(19)-R-X(20)-S-E-R-F-X(21)-X(22)-L-T-P-N-Y-N-P-D-I-I-F-K-D-E-E-N-X(23)-G-A-D-R-L-M-T-X(24)-R-C-K-X(25)-X(26)-X(27)-N-X(28)-L-A-I-S-V-M-J-X(29)-W-P-G-V-X(30)-L-R-V-T-E-G-X(31)-D-E-D-G-H-H-X(32)-X(33)-X(34)-S-L-H-Y-E-G-R-A-X(35)-D-I-T-T-S-D-R-D-X(36)-X(37)-K-Y-G-X(38)-L-X(39)-R-L-A-V-E-A-G-F-D-W-V-Y-YE-S-X(40)-X(41)-H-X(42)-H-X(43)-S-V- K-X(44)-X(45),wherein, as above, each of the degenerate positions “X” can be an aminoacid which occurs in a corresponding position in one of the wild-typeclones, and may also include amino acid residue which would beconservative substitutions, or each X can be any amino acid residue. Inan exemplary embodiment, Xaa(1) represents Gly, Ala, Val, Leu, Ile, Pro,Phe or Tyr; Xaa(2) represents Gly, Ala, Val, Leu or Ile; Xaa(3)represents Gly, Ala, Val, Leu, Ile, Lys, His or Arg; Xaa(4) representsLys, Arg or His; Xaa(5) represents Phe, Trp, Tyr or an amino acid gap;Xaa(6) represents Gly, Ala, Val, Leu, Ile or an amino acid gap; Xaa(7)represents Asn, Gln, His, Arg or Lys; Xaa(8) represents Gly, Ala, Val,Leu, Ile, Ser or Thr; Xaa(9) represents Gly, Ala, Val, Leu, Ile, Ser orThr; Xaa(10) represents Gly, Ala, Val, Leu, Ile, Ser or Thr; Xaa(11)represents Ser, Thr, Gln or Asn; Xaa(12) represents Met, Cys, Gly, Ala,Val, Leu, Ile, Ser or Thr; Xaa(13) represents Gly, Ala, Val, Leu, Ile orPro; Xaa(14) represents Arg, His or Lys; Xaa(15) represents Gly, Ala,Val, Leu, Ile, Pro, Arg, His or Lys; Xaa(16) represents Gly, Ala, Val,Leu, Ile, Phe or Tyr; Xaa(17) represents Arg, His or Lys; Xaa(18)represents Gly, Ala, Val, Leu, Ile, Ser or Thr; Xaa(19) represents Thror Ser; Xaa(20) represents Gly, Ala, Val, Leu, Ile, Asn or Gln; Xaa(21)represents Arg, His or Lys; Xaa(22) represents Asp or Glu; Xaa(23)represents Ser or Thr; Xaa(24) represents Glu, Asp, Gln or Asn; Xaa(25)represents Glu or Asp; Xaa(26) represents Arg, His or Lys; Xaa(27)represents Gly, Ala, Val, Leu or Ile; Xaa(28) represents Gly, Ala, Val,Leu, Ile, Thr or Ser; Xaa(29) represents Met, Cys, Gln, Asn, Arg, Lys orHis; Xaa(30) represents Arg, His or Lys; Xaa(31) represents Trp, Phe,Tyr, Arg, His or Lys; Xaa(32) represents Gly, Ala, Val, Leu, Ile, Ser,Thr, Tyr or Phe; Xaa(33) represents Gln, Asn, Asp or Glu; Xaa(34)represents Asp or Glu; Xaa(35) represents Gly, Ala, Val, Leu, or Ile;Xaa(36) represents Arg, His or Lys; Xaa(37) represents Asn, Gln, Thr orSer; Xaa(38) represents Gly, Ala, Val, Leu, Ile, Ser, Thr, Met or Cys;Xaa(39) represents Gly, Ala, Val, Leu, Ile, Thr or Ser; Xaa(40)represents Arg, His or Lys; Xaa(41) represents Asn, Gln, Gly, Ala, Val,Leu or Ile; Xaa(42) represents Gly, Ala, Val, Leu or Ile; Xaa(43)represents Gly, Ala, Val, Leu, Ile, Ser, Thr or Cys; Xaa(44) representsGly, Ala, Val, Leu, Ile, Thr or Ser; and Xaa(45) represents Asp or Glu.

There are many ways by which the library of potential hedgehog homologscan be generated from a degenerate oligonucleotide sequence. Chemicalsynthesis of a degenerate gene sequence can be carried out in anautomatic DNA synthesizer, and the synthetic genes then ligated into anappropriate expression vector. The purpose of a degenerate set of genesis to provide, in one mixture, all of the sequences encoding the desiredset of potential hedgehog sequences. The synthesis of degenerateoligonucleotides is well known in the art (see for example, Narang, S A(1983) Tetrahedron 39:3; Itakura et al. (1981) Recombinant DNA, Proc 3rdCleveland Sympos. Macromolecules, ed. A G Walton, Amsterdam: Elsevierpp273–289; Itakura et al. (1984) Annu. Rev. Biochem. 53:323; Itakura etal. (1984) Science 198:1056; Ike et al. (1983) Nucleic Acid Res. 11:477.Such techniques have been employed in the directed evolution of otherproteins (see, for example, Scott et al. (1990) Science 249:386–390;Roberts et al. (1992) PNAS 89:2429–2433; Devlin et al. (1990) Science249: 404–406; Cwirla et al. (1990) PNAS 87: 6378–6382; as well as U.S.Pat. Nos. 5,223,409, 5,198,346, and 5,096,815).

A wide range of techniques are known in the art for screening geneproducts of combinatorial libraries made by point mutations, and forscreening cDNA libraries for gene products having a certain property.Such techniques will be generally adaptable for rapid screening of thegene libraries generated by the combinatorial mutagenesis of hedgehoghomologs. The most widely used techniques for screening large genelibraries typically comprises cloning the gene library into replicableexpression vectors, transforming appropriate cells with the resultinglibrary of vectors, and expressing the combinatorial genes underconditions in which detection of a desired activity facilitatesrelatively easy isolation of the vector encoding the gene whose productwas detected. Each of the illustrative assays described below areamenable to high through-put analysis as necessary to screen largenumbers of degenerate hedgehog sequences created by combinatorialmutagenesis techniques.

In one embodiment, the combinatorial library is designed to be secreted(e.g. the polypeptides of the library all include a signal sequence butno transmembrane or cytoplasmic domains), and is used to transfect aeukaryotic cell that can be co-cultured with neuronal cells. Afunctional hedgehog protein secreted by the cells expressing thecombinatorial library will diffuse to neighboring neuronal cells andinduce a particular biological response, such as protection against celldeath when treated with MPTP. The pattern of protection will resemble agradient function, and will allow the isolation (generally after severalrepetitive rounds of selection) of cells producing hedgehog homologsactive as neuroprotective agents with respect to the target neuronalcells

To illustrate, target neuronal cells are cultured in 24-well microtitreplates. Other eukaryotic cells are transfected with the combinatorialhedgehog gene library and cultured in cell culture inserts (e.g.Collaborative Biomedical Products, Catalog #40446) that are able to fitinto the wells of the microtitre plate. The cell culture inserts areplaced in the wells such that recombinant hedgehog homologs secreted bythe cells in the insert can diffuse through the porous bottom of theinsert and contact the target cells in the microtitre plate wells. Aftera period of time sufficient for functional forms of a hedgehog proteinto produce a measurable response in the target cells, such asneuroprotection, the inserts are removed and the effect of the varianthedgehog proteins on the target cells determined. Cells from the insertscorresponding to wells which score positive for activity can be splitand re-cultured on several inserts, the process being repeated until theactive clones are identified.

In yet another screening assay, the candidate hedgehog gene products aredisplayed on the surface of a cell or viral particle, and the ability ofparticular cells or viral particles to associate with a hedgehog-bindingmoiety (such as the patched protein or other hedgehog receptor) via thisgene product is detected in a “panning assay”. Such panning steps can becarried out on cells cultured from embryos. For instance, the genelibrary can be cloned into the gene for a surface membrane protein of abacterial cell, and the resulting fusion protein detected by panning(Ladner et al., WO 88/06630; Fuchs et al. (1991) Bio/Technology9:1370–1371; and Goward et al. (1992) TIBS 18:136–140). In a similarfashion, fluorescently labeled molecules which bind hedgehog can be usedto score for potentially functional hedgehog homologs. Cells can bevisually inspected and separated under a fluorescence microscope, or,where the morphology of the cell permits, separated by afluorescence-activated cell sorter.

In an alternate embodiment, the gene library is expressed as a fusionprotein on the surface of a viral particle. For instance, in thefilamentous phage system, foreign peptide sequences can be expressed onthe surface of infectious phage, thereby conferring two significantbenefits. First, since these phage can be applied to affinity matricesat very high concentrations, large number of phage can be screened atone time. Second, since each infectious phage displays the combinatorialgene product on its surface, if a particular phage is recovered from anaffinity matrix in low yield, the phage can be amplified by anotherround of infection. The group of almost identical E.coli filamentousphages M13, fd, and fl are most often used in phage display libraries,as either of the phage gIII or gVIII coat proteins can be used togenerate fusion proteins without disrupting the ultimate packaging ofthe viral particle (Ladner et al. PCT publication WO 90/02909; Garrardet al., PCT publication WO 92/09690; Marks et al. (1992) J. Biol. Chem.267:16007–16010; Griffths et al. (1993) EMBO J. 12:725–734; Clackson etal. (1991) Nature 352:624–628; and Barbas et al. (1992) PNAS89:4457–4461).

In an illustrative embodiment, the recombinant phage antibody system(RPAS, Pharamacia Catalog number 27-9400-01) can be easily modified foruse in expressing and screening hedgehog combinatorial libraries. Forinstance, the pCANTAB 5 phagemid of the RPAS kit contains the gene whichencodes the phage gIII coat protein. The hedgehog combinatorial genelibrary can be cloned into the phagemid adjacent to the gIII signalsequence such that it will be expressed as a gIII fusion protein. Afterligation, the phagemid is used to transform competent E. coli TG1 cells.Transformed cells are subsequently infected with M13KO7 helper phage torescue the phagemid and its candidate hedgehog gene insert. Theresulting recombinant phage contain phagemid DNA encoding a specificcandidate hedgehog, and display one or more copies of the correspondingfusion coat protein. The phage-displayed candidate hedgehog proteinswhich are capable of binding an hedgehog receptor are selected orenriched by panning. For instance, the phage library can be applied tocells which express the patched protein and unbound phage washed awayfrom the cells. The bound phage is then isolated, and if the recombinantphage express at least one copy of the wild type gIII coat protein, theywill retain their ability to infect E. coli. Thus, successive rounds ofreinfection of E. coli, and panning will greatly enrich for hedgehoghomologs, which can then be screened for further biological activitiesin order to differentiate agonists and antagonists.

Combinatorial mutagenesis has a potential to generate very largelibraries of mutant proteins, e.g., in the order of 10²⁶ molecules.Combinatorial libraries of this size may be technically challenging toscreen even with high throughput screening assays such as phage display.To overcome this problem, a new technique has been developed recently,recrusive ensemble mutagenesis (REM), which allows one to avoid the veryhigh proportion of non-functional proteins in a random library andsimply enhances the frequency of functional proteins, thus decreasingthe complexity required to achieve a useful sampling of sequence space.REM is an algorithm which enhances the frequency of functional mutantsin a library when an appropriate selection or screening method isemployed (Arkin and Yourvan, 1992, PNAS USA 89:7811–7815; Yourvan etal., 1992, Parallel Problem Solving from Nature, 2., In Maenner andManderick, eds., Elsevir Publishing Co., Amsterdam, pp. 401–410;Delgrave et al., 1993, Protein Engineering 6(3):327–331).

The invention also provides for reduction of the hedgehog protein togenerate mimetics, e.g. peptide or non-peptide agents, which are able tomimic the neuroprotective activity of a naturally-occurring hedgehogpolypeptide. Thus, such mutagenic techniques as described above are alsouseful to map the determinants of the hedgehog proteins whichparticipate in protein-protein interactions involved in, for example,binding of the subject hedgehog polypeptide to other extracellularmatrix components such as its receptor(s). To illustrate, the criticalresidues of a subject hedgehog polypeptide which are involved inmolecular recognition of an hedgehog receptor such as patched can bedetermined and used to generate hedgehog-derived peptidomimetics whichcompetitively bind with that moiety. By employing, for example, scanningmutagenesis to map the amino acid residues of each of the subjecthedgehog proteins which are involved in binding other extracellularproteins, peptidomimetic compounds can be generated which mimic thoseresidues of the hedgehog protein which facilitate the interaction. Afterdistinguishing between agonist and antagonists, such agonistic mimeticsmay be used to mimic the normal function of a hedgehog protein astrophic for dopaminergic and GABAergic neurons. For instance,non-hydrolyzable peptide analogs of such residues can be generated usingbenzodiazepine (e.g., see Freidinger et al. in Peptides: Chemistry andBiology, G. R. Marshall ed., ESCOM Publisher: Leiden, Netherlands,1988), azepine (e.g., see Huffman et al. in Peptides: Chemistry andBiology, G. R. Marshall ed., ESCOM Publisher: Leiden, Netherlands,1988), substituted gama lactam rings (Garvey et al. in Peptides:Chemistry and Biology, G. R. Marshall ed., ESCOM Publisher: Leiden,Netherlands, 1988), keto-methylene pseudopeptides (Ewenson et al. (1986)J Med Chem 29:295; and Ewenson et al. in Peptides: Structure andFunction (Proceedings of the 9th American Peptide Symposium) PierceChemical Co. Rockland, Ill., 1985), β-turn dipeptide cores (Nagai et al.(1985) Tetrahedron Lett 26:647; and Sato et al. (1986) J Chem Soc PerkinTrans 1:1231), and β-aminoalcohols (Gordon et al. (1985) Biochem BiophysRes Commun 126:419; and Dann et al. (1986) Biochem Biophys Res Commun134:71).

Recombinantly produced forms of the hedgehog proteins can be producedusing, e.g, expression vectors containing a nucleic acid encoding ahedgehog polypeptide, operably linked to at least one transcriptionalregulatory sequence. Operably linked is intended to mean that thenucleotide sequence is linked to a regulatory sequence in a manner whichallows expression of the nucleotide sequence. Regulatory sequences areart-recognized and are selected to direct expression of a hedgehogpolypeptide. Accordingly, the term transcriptional regulatory sequenceincludes promoters, enhancers and other expression control elements.Such regulatory sequences are described in Goeddel; Gene ExpressionTechnology: Methods in Enzymology 185, Academic Press, San Diego, Calif.(1990). For instance, any of a wide variety of expression controlsequences, sequences that control the expression of a DNA sequence whenoperatively linked to it, may be used in these vectors to express DNAsequences encoding hedgehog polypeptide. Such useful expression controlsequences, include, for example, a viral LTR, such as the LTR of theMoloney murine leukemia virus, the early and late promoters of SV40,adenovirus or cytomegalovirus immediate early promoter, the lac system,the trp system, the TAC or TRC system, T7 promoter whose expression isdirected by T7 RNA polymerase, the major operator and promoter regionsof phage λ, the control regions for fd coat protein, the promoter for3-phosphoglycerate kinase or other glycolytic enzymes, the promoters ofacid phosphatase, e.g., Pho5, the promoters of the yeast α-matingfactors, the polyhedron promoter of the baculovirus system and othersequences known to control the expression of genes of prokaryotic oreukaryotic cells or their viruses, and various combinations thereof. Itshould be understood that the design of the expression vector may dependon such factors as the choice of the host cell to be transformed and/orthe type of protein desired to be expressed. Moreover, the vector's copynumber, the ability to control that copy number and the expression ofany other proteins encoded by the vector, such as antibiotic markers,should also be considered.

In addition to providing a ready source of hedgehog polypeptides forpurification, the gene constructs of the present invention can also beused as a part of a gene therapy protocol to deliver nucleic acidsencoding either a neuroprotective form of a hedgehog polypeptide. Thus,another aspect of the invention features expression vectors for in vivotransfection of a hedgehog polypeptide in particular cell types so as tocause ectopic expression of a hedgehog polypeptide in neuronal tissue.

Formulations of such expression constructs may be administered in anybiologically effective carrier, e.g. any formulation or compositioncapable of effectively delivering the recombinant gene to cells in vivo.Approaches include insertion of the hedgehog coding sequence in viralvectors including recombinant retroviruses, adenovirus, adeno-associatedvirus, and herpes simplex virus-1, or recombinant bacterial oreukaryotic plasmids. Viral vectors transfect cells directly; plasmid DNAcan be delivered with the help of, for example, cationic liposomes(lipofectin) or derivatized (e.g. antibody conjugated), polylysineconjugates, gramacidin S, artificial viral envelopes or other suchintracellular carriers, as well as direct injection of the geneconstruct or CaPO₄ precipitation carried out in vivo. It will beappreciated that because transduction of appropriate target cellsrepresents the critical first step in gene therapy, choice of theparticular gene delivery system will depend on such factors as thephenotype of the intended target and the route of administration, e.g.locally or systemically. Furthermore, it will be recognized that theparticular gene construct provided for in vivo transduction of hedgehogexpression are also useful for in vitro transduction of cells, such asfor use in the ex vivo tissue culture systems described below.

A preferred approach for in vivo introduction of nucleic acid into acell is by use of a viral vector containing nucleic acid, e.g. a cDNA,encoding the particular form of the hedgehog polypeptide desired.Infection of cells with a viral vector has the advantage that a largeproportion of the targeted cells can receive the nucleic acid.Additionally, molecules encoded within the viral vector, e.g., by a cDNAcontained in the viral vector, are expressed efficiently in cells whichhave taken up viral vector nucleic acid.

Retrovirus vectors and adeno-associated virus vectors are generallyunderstood to be the recombinant gene delivery system of choice for thetransfer of exogenous genes in vivo, particularly into humans. Thesevectors provide efficient delivery of genes into cells, and thetransferred nucleic acids are stably integrated into the chromosomal DNAof the host. A major prerequisite for the use of retroviruses is toensure the safety of their use, particularly with regard to thepossibility of the spread of wild-type virus in the cell population. Thedevelopment of specialized cell lines (termed “packaging cells”) whichproduce only replication-defective retroviruses has increased theutility of retroviruses for gene therapy, and defective retroviruses arewell characterized for use in gene transfer for gene therapy purposes(for a review see Miller, A. D. (1990) Blood 76:271). Thus, recombinantretrovirus can be constructed in which part of the retroviral codingsequence (gag, pol, env) has been replaced by nucleic acid encoding ahedgehog polypeptide and renders the retrovirus replication defective.The replication defective retrovirus is then packaged into virions whichcan be used to infect a target cell through the use of a helper virus bystandard techniques. Protocols for producing recombinant retrovirusesand for infecting cells in vitro or in vivo with such viruses can befound in Current Protocols in Molecular Biology, Ausubel, F. M. et al.(eds.) Greene Publishing Associates, (1989), Sections 9.10–9.14 andother standard laboratory manuals. Examples of suitable retrovirusesinclude pLJ, pZIP, pWE and pEM which are well known to those skilled inthe art. Examples of suitable packaging virus lines for preparing bothecotropic and amphotropic retroviral systems include Crip, Cre, 2 andAm. Retroviruses have been used to introduce a variety of genes intomany different cell types, including neuronal cells, in vitro and/or invivo (see for example Eglitis, et al. (1985) Science 230:1395–1398;Danos and Mulligan (1988) Proc. Natl. Acad. Sci. USA 85:6460–6464;Wilson et al. (1988) Proc. Natl. Acad. Sci. USA 85:3014–3018; Armentanoet al. (1990) Proc. Natl. Acad. Sci. USA 87:6141–6145; Huber et al.(1991) Proc. Natl. Acad. Sci. USA 88:8039–8043; Ferry et al. (1991)Proc. Natl. Acad. Sci. USA 88:8377–8381; Chowdhury et al. (1991) Science254:1802–1805; van Beusechem et al. (1992) Proc. Natl. Acad. Sci. USA89:7640–7644; Kay et al. (1992) Human Gene Therapy 3:641–647; Dai et al.(1992) Proc. Natl. Acad. Sci. USA 89:10892–10895; Hwu et al. (1993) J.Immunol. 150:4104–4115; U.S. Pat. No. 4,868,116; U.S. Pat. No.4,980,286; PCT Application WO 89/07136; PCT Application WO 89/02468; PCTApplication WO 89/05345; and PCT Application WO 92/07573).

Furthermore, it has been shown that it is possible to limit theinfection spectrum of retroviruses and consequently of retroviral-basedvectors, by modifying the viral packaging proteins on the surface of theviral particle (see, for example PCT publications WO93/25234 andWO94/06920). For instance, strategies for the modification of theinfection spectrum of retroviral vectors include: coupling antibodiesspecific for cell surface antigens to the viral env protein (Roux et al.(1989) PNAS 86:9079–9083; Julan et al. (1992) J. Gen Virol 73:3251–3255;and Goud et al. (1983) Virology 163:251–254); or coupling cell surfacereceptor ligands to the viral env proteins (Neda et al. (1991) J BiolChem 266:14143–14146). Coupling can be in the form of the chemicalcross-linking with a protein or other variety (e.g. lactose to convertthe env protein to an asialoglycoprotein), as well as by generatingfusion proteins (e.g. single-chain antibody/env fusion proteins). Thistechnique, while useful to limit or otherwise direct the infection tocertain tissue types, can also be used to convert an ecotropic vector into an amphotropic vector.

Moreover, use of retroviral gene delivery can be further enhanced by theuse of tissue- or cell-specific transcriptional regulatory sequenceswhich control expression of the hedgehog gene of the retroviral vector.

Another viral gene delivery system useful in the present method utilizesadenovirus-derived vectors. The genome of an adenovirus can bemanipulated such that it encodes and expresses a gene product ofinterest but is inactivated in terms of its ability to replicate in anormal lytic viral life cycle. See for example Berkner et al. (1988)BioTechniques 6:616; Rosenfeld et al. (1991) Science 252:431–434; andRosenfeld et al. (1992) Cell 68:143–155. Suitable adenoviral vectorsderived from the adenovirus strain Ad type 5 d1324 or other strains ofadenovirus (e.g., Ad2, Ad3, Ad7 etc.) are well known to those skilled inthe art. Recombinant adenoviruses can be advantageous in certaincircumstances in that they can be used to infect a wide variety of celltypes, including neuronal cells (Rosenfeld et al. (1992) cited supra).

Furthermore, the virus particle is relatively stable and amenable topurification and concentration, and as above, can be modified so as toaffect the spectrum of infectivity. Additionally, introduced adenoviralDNA (and foreign DNA contained therein) is not integrated into thegenome of a host cell but remains episomal, thereby avoiding potentialproblems that can occur as a result of insertional mutagenesis insituations where introduced DNA becomes integrated into the host genome(e.g., retroviral DNA). Moreover, the carrying capacity of theadenoviral genome for foreign DNA is large (up to 8 kilobases) relativeto other gene delivery vectors (Berkner et al. cited supra; Haj-Ahmandand Graham (1986) J. Virol. 57:267). Most replication-defectiveadenoviral vectors currently in use and therefore favored by the presentinvention are deleted for all or parts of the viral E1 and E3 genes butretain as much as 80% of the adenoviral genetic material (see, e.g.,Jones et al. (1979) Cell 16:683; Berkner et al., supra; and Graham etal. in Methods in Molecular Biology, E. J. Murray, Ed. (Humana, Clifton,N.J., 1991) vol. 7. pp. 109–127). Expression of the inserted hedgehoggene can be under control of, for example, the E1A promoter, the majorlate promoter (MLP) and associated leader sequences, the E3 promoter, orexogenously added promoter sequences.

In addition to viral transfer methods, such as those illustrated above,non-viral methods can also be employed to cause expression of a hedgehogpolypeptide in the tissue of an animal. Most nonviral methods of genetransfer rely on normal mechanisms used by mammalian cells for theuptake and intracellular transport of macromolecules. In preferredembodiments, non-viral gene delivery systems of the present inventionrely on endocytic pathways for the uptake of the hedgehog polypeptidegene by the targeted cell. Exemplary gene delivery systems of this typeinclude liposomal derived systems, poly-lysine conjugates, andartificial viral envelopes.

In clinical settings, the gene delivery systems for the therapeutichedgehog gene can be introduced into a patient by any of a number ofmethods, each of which is familiar in the art. For instance, apharmaceutical preparation of the gene delivery system can be introducedsystemically, e.g. by intravenous injection, and specific transductionof the protein in the target cells occurs predominantly from specificityof transfection provided by the gene delivery vehicle, cell-type ortissue-type expression due to the transcriptional regulatory sequencescontrolling expression of the receptor gene, or a combination thereof.In other embodiments, initial delivery of the recombinant gene is morelimited with introduction into the animal being quite localized. Forexample, the gene delivery vehicle can be introduced by catheter (seeU.S. Pat. No. 5,328,470) or by stereotactic injection (e.g. Chen et al.(1994) PNAS 91: 3054–3057). A hedgehog expression construct can bedelivered in a gene therapy construct to dermal cells by, e.g.,electroporation using techniques described, for example, by Dev et al.((1994) Cancer Treat Rev 20:105–115).

The pharmaceutical preparation of the gene therapy construct can consistessentially of the gene delivery system in an acceptable diluent, or cancomprise a slow release matrix in which the gene delivery vehicle isimbedded. Alternatively, where the complete gene delivery system can beproduced intact from recombinant cells, e.g. retroviral vectors, thepharmaceutical preparation can comprise one or more cells which producethe gene delivery system.

In yet another embodiment, the hedgehog or ptc therapeutic can be a“gene activation”construct which, by homologous recombination with agenomic DNA, alters the transcriptional regulatory sequences of anendogenous gene. For instance, the gene activation construct can replacethe endogenous promoter of a hedgehog gene with a heterologous promoter,e.g., one which causes constitutive expression of the hedgehog gene orwhich causes inducible expression of the gene under conditions differentfrom the normal expression pattern of the gene. Other genes in thepatched signaling pathway can be similarly targeted. A variety ofdifferent formats for the gene activation constructs are available. See,for example, the Transkaryotic Therapies, Inc PCT publicationsWO93/09222, WO95/31560, WO96/29411, WO95/31560 and WO94/12650.

In preferred embodiments, the nucleotide sequence used as the geneactivation construct can be comprised of (1) DNA from some portion ofthe endogenous hedgehog gene (exon sequence, intron sequence, promotersequences, etc.) which direct recombination and (2) heterologoustranscriptional regulatory sequence(s) which is to be operably linked tothe coding sequence for the genomic hedgehog gene upon recombination ofthe gene activation construct. For use in generating cultures ofhedgehog producing cells, the construct may further include a reportergene to detect the presence of the knockout construct in the cell.

The gene activation construct is inserted into a cell, and integrateswith the genomic DNA of the cell in such a position so as to provide theheterologous regulatory sequences in operative association with thenative hedgehog gene. Such insertion occurs by homologous recombination,i.e., recombination regions of the activation construct that arehomologous to the endogenous hedgehog gene sequence hybridize to thegenomic DNA and recombine with the genomic sequences so that theconstruct is incorporated into the corresponding position of the genomicDNA.

The terms “recombination region” or “targeting sequence” refer to asegment (i.e., a portion) of a gene activation construct having asequence that is substantially identical to or substantiallycomplementary to a genomic gene sequence, e.g., including 5′ flankingsequences of the genomic gene, and can facilitate homologousrecombination between the genomic sequence and the targeting transgeneconstruct.

As used herein, the term “replacement region” refers to a portion of anactivation construct which becomes integrated into an endogenouschromosomal location following homologous recombination between arecombination region and a genomic sequence.

The heterologous regulatory sequences, e.g., which are provided in thereplacement region, can include one or more of a variety elements,including: promoters (such as constitutive or inducible promoters),enhancers, negative regulatory elements, locus control regions,transcription factor binding sites, or combinations thereof.Promoters/enhancers which may be used to control the expression of thetargeted gene in vivo include, but are not limited to, thecytomegalovirus (CMV) promoter/enhancer (Karasuyama et al., 1989, J.Exp. Med., 169:13), the human β-actin promoter (Gunning et al. (1987)PNAS 84:4831–4835), the glucocorticoid-inducible promoter present in themouse mammary tumor virus long terminal repeat (MMTV LTR) (Klessig etal. (1984) Mol. Cell Biol. 4:1354–1362), the long terminal repeatsequences of Moloney murine leukemia virus (MuLV LTR) (Weiss et al.(1985) RNA Tumor Viruses, Cold Spring Harbor Laboratory, Cold SpringHarbor, N.Y.), the SV40 early or late region promoter (Bernoist et al.(1981) Nature 290:304–310; Templeton et al. (1984) Mol. Cell Biol.,4:817; and Sprague et al. (1983) J. Virol., 45:773), the promotercontained in the 3′ long terminal repeat of Rous sarcoma virus (RSV)(Yamamoto et al., 1980, Cell, 22:787–797), the herpes simplex virus(HSV) thymidine kinase promoter/enhancer (Wagner et al. (1981) PNAS82:3567–71), and the herpes simplex virus LAT promoter (Wolfe et al.(1992) Nature Genetics, 1:379–384).

In an exemplary embodiment, portions of the 5′ flanking region of thehuman Shh gene are amplified using primers which add restriction sites,to generate the following fragments

(SEQ ID NO: 23)5′-gcgcgcttcgaaGCGAGGCAGCCAGCGAGGGAGAGAGCGAGCGGGCGAGCCGGAGCGAGGAAatcgatgcgcgc(primer 1) (SEQ ID NO: 24)5′-gcgcgcagatctGGGAAAGCGCAAGAGAGAGCGCACACGCACACACCCGCCGCGCGCACTCGggatccgcgcgc(primer 2)As illustrated, primer 1 includes a 5′ non-coding region of the humanShh gene and is flanked by an AsulI and ClaI restriction sites. Primer 2includes a portion of the 5′ non-coding region immediately 3′ to thatpresent in primer 1. The hedgehog gene sequence is flanked by XhoII andBamHI restriction sites. The purified amplimers are cut with each of theenzymes as appropriate.

The vector pcDNA1.1 (Invitrogen) includes a CMV promoter. The plasmid iscut with with AsulI, which cleaves just 3′ to the CMV promoter sequence.The AsulI/ClaI fragment of primer 1 is ligated to the AsulI cleavagesite of the pcDNA vector. The ClaI/AsulI ligation destroys the AsulIsite at the 3′ end of a properly inserted primer 1.

The vector is then cut with BamHI, and an XhoII/BamHI fragment of primer2 is ligated to the BamHI cleavage site. As above, the BamHI/XhoIIligation destroys the BamHI site at the 5′ end of a properly insertedprimer 2.

Individual colonies are selected, cut with Asull and BamHI, and the sizeof the AsuII/BamHI fragment determined. Colonies in which both theprimer 1 and primer 2 sequences are correctly inserted are furtheramplified, an cut with Asull and BamHI to produce the gene activationconstruct

(SEQ ID NO: 25) cgaagcgaggcagccagcgagggagagagcgagcgggcgagccggagcgaggaaATCGAAGGTTCGAATCCTTCCCCCACCACCATCACTTTCAAAAGTCCGAAAGAATCTGCTCCCTGCTTGTGTGTTGGAGGTCGCTGAGTAGTGCGCGAGTAATTTAAGCTACAACAAGGCAAGGCTTGACCGACAATTGCATGAAGAATCTGCTTAGGGTTAGGCGTTTTGCGCTGCTTCGCGATGTACGGGCCAGATATACGCGTTGACATTGATTATTGACTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGACTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTCTCTGGCTAACTAGAGAACCCACTGCTTACTGGCTTATCGAAATTAATACGACTCACTATAGGGAGACCCAAGCTTGGTACCGAGCTCGGATCgatctgggaaagcgcaagagagagcgcacacgcacacacccgccgcgcgcactcggIn this construct, the flanking primer 1 and primer 2 sequences providethe recombination region which permits the insertion of the CMV promoterin front of the coding sequence for the human Shh gene. Otherheterologous promoters (or other transcriptional regulatory sequences)can be inserted in a genomic hedgehog gene by a similar method.

In still other embodiments, the replacement region merely deletes anegative transcriptional control element of the native gene, e.g., toactivate expression, or ablates a positive control element, e.g., toinhibit expression of the targeted gene.

V. Exemplary ptc Therapeutic Compounds

In another embodiment, the subject method is carried out using a ptctherapeutic composition. Such compositions can be generated with, forexample, compounds which bind to patched and alter its signaltransduction activity, compounds which alter the binding and/orenzymatic activity of a protein (e.g., intracellular) involved inpatched signal pathway, and compounds which alter the level ofexpression of a hedgehog protein, a patched protein or a proteininvolved in the intracellular signal transduction pathway of patched.

The availability of purified and recombinant hedgehog polypeptidesfacilitates the generation of assay systems which can be used to screenfor drugs, such as small organic molecules, which are either agonists orantagonists of the normal cellular function of a hedgehog and/or patchedprotein, particularly in their role in the pathogenesis of neuronal celldeath. In one embodiment, the assay evaluates the ability of a compoundto modulate binding between a hedgehog polypeptide and a hedgehogreceptor such as patched. In other embodiments, the assay merely scoresfor the ability of a test compound to alter the signal transductionactivity of the patched protein. In this manner, a variety of hedgehogand/or ptc therapeutics, which will include ones with neuroprotectiveactivity, can be identified. A variety of assay formats will sufficeand, in light of the present disclosure, will be comprehended by skilledartisan.

In many drug screening programs which test libraries of compounds andnatural extracts, high throughput assays are desirable in order tomaximize the number of compounds surveyed in a given period of time.Assays which are performed in cell-free systems, such as may be derivedwith purified or semi-purified proteins, are often preferred as“primary” screens in that they can be generated to permit rapiddevelopment and relatively easy detection of an alteration in amolecular target which is mediated by a test compound. Moreover, theeffects of cellular toxicity and/or bioavailability of the test compoundcan be generally ignored in the in vitro system, the assay instead beingfocused primarily on the effect of the drug on the molecular target asmay be manifest in an alteration of binding affinity with receptorproteins.

Accordingly, in an exemplary screening assay for ptc therapeutics, thecompound of interest is contacted with a mixture including a hedgehogreceptor protein (e.g., a cell expressing the patched receptor) and ahedgehog protein under conditions in which it is ordinarily capable ofbinding the hedgehog protein. To the mixture is then added a compositioncontaining a test compound. Detection and quantification ofreceptor/hedgehog complexes provides a means for determining the testcompound's efficacy at inhibiting (or potentiating) complex formationbetween the receptor protein and the hedgehog polypeptide. Moreover, acontrol assay can also be performed to provide a baseline forcomparison. In the control assay, isolated and purified hedgehogpolypeptide is added to the receptor protein, and the formation ofreceptor/hedgehog complex is quantitated in the absence of the testcompound.

In other embodiments, a ptc therapeutic of the present invention is onewhich disrupts the association of patched with smoothened.

Agonist and antagonists of neuroprotection can be distinguished, and theefficacy of the compound can be assessed, by subsequent testing withneuronal cells.

In an illustrative embodiment, the polypeptide utilized as a hedgehogreceptor can be generated from the patched protein. Accordingly, anexemplary screening assay includes all or a suitable portion of thepatched protein which can be obtained from, for example, the humanpatched gene (GenBank U43148) or other vertebrate sources (see GenBankAccession numbers U40074 for chicken patched and U46155 for mousepatched), as well as from drosophila (GenBank Accession number M28999)or other invertebrate sources. The patched protein can be provided inthe screening assay as a whole protein (preferably expressed on thesurface of a cell), or alternatively as a fragment of the full lengthprotein which binds to hedgehog polypeptides, e.g., as one or both ofthe substantial extracellular domains (e.g. corresponding to residuesAsn120-Ser438 and/or Arg770-Trp1027 of the human patched protein). Forinstance, the patched protein can be provided in soluble form, as forexample a preparation of one of the extracellular domains, or apreparation of both of the extracellular domains which are covalentlyconnected by an unstructured linker (see, for example, Huston et al.(1988) PNAS 85:4879; and U.S. Pat. No. 5,091,513). In other embodiments,the protein can be provided as part of a liposomal preparation orexpressed on the surface of a cell. The patched protein can derived froma recombinant gene, e.g., being ectopically expressed in a heterologouscell. For instance, the protein can be expressed on oocytes, mammaliancells (e.g., COS, CHO, 3T3 or the like), or yeast cells by standardrecombinant DNA techniques. These recombinant cells can be used forreceptor binding, signal transduction or gene expression assays. Marigoet al. (1996) Development 122:1225–1233 illustrates a binding assay ofhuman hedgehog to chick patched protein ectopically expressed in Xenopuslaevis oocytes. The assay system of Marigo et al. can be adapted to thepresent drug screening assays. As illustrated in that reference, Shhbinds to the patched protein in a selective, saturable, dose-dependentmanner, thus demonstrating that patched is a receptor for Shh.

Complex formation between the hedgehog polypeptide and a hedgehogreceptor may be detected by a variety of techniques. For instance,modulation of the formation of complexes can be quantitated using, forexample, detectably labelled proteins such as radiolabelled,fluorescently labelled, or enzymatically labelled hedgehog polypeptides,by immunoassay, or by chromatographic detection.

Typically, for cell-free assays, it will be desirable to immobilizeeither the hedgehog receptor or the hedgehog polypeptide to facilitateseparation of receptor/hedgehog complexes from uncomplexed forms of oneof the proteins, as well as to accommodate automation of the assay. Inone embodiment, a fusion protein can be provided which adds a domainthat allows the protein to be bound to a matrix. For example,glutathione-S-transferase/receptor (GST/receptor) fusion proteins can beadsorbed onto glutathione sepharose beads (Sigma Chemical, St. Louis,Mo.) or glutathione derivatized microtitre plates, which are thencombined with the hedgehog polypeptide, e.g. an ³⁵S-labeled hedgehogpolypeptide, and the test compound and incubated under conditionsconducive to complex formation, e.g. at physiological conditions forsalt and pH, though slightly more stringent conditions may be desired.Following incubation, the beads are washed to remove any unboundhedgehog polypeptide, and the matrix bead-bound radiolabel determineddirectly (e.g. beads placed in scintillant), or in the supernatant afterthe receptor/hedgehog complexes are dissociated. Alternatively, thecomplexes can be dissociated from the bead, separated by SDS-PAGE gel,and the level of hedgehog polypeptide found in the bead fractionquantitated from the gel using standard electrophoretic techniques.

Other techniques for immobilizing proteins on matrices are alsoavailable for use in the subject assay. For instance, soluble portionsof the hedgehog receptor protein can be immobilized utilizingconjugation of biotin and streptavidin. For instance, biotinylatedreceptor molecules can be prepared frombiotin-NHS(N-hydroxy-succinimide) using techniques well known in the art(e.g., biotinylation kit, Pierce Chemicals, Rockford, Ill.), andimmobilized in the wells of streptavidin-coated 96 well plates (PierceChemical). Alternatively, antibodies reactive with the hedgehog receptorbut which do not interfere with hedgehog binding can be derivatized tothe wells of the plate, and the receptor trapped in the wells byantibody conjugation. As above, preparations of a hedgehog polypeptideand a test compound are incubated in the receptor-presenting wells ofthe plate, and the amount of receptor/hedgehog complex trapped in thewell can be quantitated. Exemplary methods for detecting such complexes,in addition to those described above for the GST-immobilized complexes,include immunodetection of complexes using antibodies reactive with thehedgehog polypeptide, or which are reactive with the receptor proteinand compete for binding with the hedgehog polypeptide; as well asenzyme-linked assays which rely on detecting an enzymatic activityassociated with the hedgehog polypeptide. In the instance of the latter,the enzyme can be chemically conjugated or provided as a fusion proteinwith the hedgehog polypeptide. To illustrate, the hedgehog polypeptidecan be chemically cross-linked or genetically fused with alkalinephosphatase, and the amount of hedgehog polypeptide trapped in thecomplex can be assessed with a chromogenic substrate of the enzyme, e.g.paranitrophenylphosphate. Likewise, a fusion protein comprising thehedgehog polypeptide and glutathione-S-transferase can be provided, andcomplex formation quantitated by detecting the GST activity using1-chloro-2,4-dinitrobenzene (Habig et al (1974) J Biol Chem 249:7130).

For processes which rely on immunodetection for quantitating one of theproteins trapped in the complex, antibodies against the protein, such asthe anti-hedgehog antibodies described herein, can be used.Alternatively, the protein to be detected in the complex can be “epitopetagged” in the form of a fusion protein which includes, in addition tothe hedgehog polypeptide or hedgehog receptor sequence, a secondpolypeptide for which antibodies are readily available (e.g. fromcommercial sources). For instance, the GST fusion proteins describedabove can also be used for quantification of binding using antibodiesagainst the GST moiety. Other useful epitope tags include myc-epitopes(e.g., see Ellison et al. (1991) J Biol Chem 266:21150–21157) whichincludes a 10-residue sequence from c-myc, as well as the pFLAG system(International Biotechnologies, Inc.) or the pEZZ-protein A system(Pharamacia, N.J.).

Where the desired portion of the hedgehog receptor (or other hedgehogbinding molecule) cannot be provided in soluble form, liposomal vesiclescan be used to provide manipulatable and isolatable sources of thereceptor. For example, both authentic and recombinant forms of thepatched protein can be reconstituted in artificial lipid vesicles (e.g.phosphatidylcholine liposomes) or in cell membrane-derived vesicles(see, for example, Bear et al. (1992) Cell 68:809–818; Newton et al.(1983) Biochemistry 22:6110–6117; and Reber et al. (1987) J Biol Chem262:11369–11374).

In addition to cell-free assays, such as described above, the readilyavailable source of hedgehog proteins provided by the art alsofacilitates the generation of cell-based assays for identifying smallmolecule agonists of the neuroprotective activity of wild-type hedgehogproteins. Analogous to the cell-based assays described above forscreening combinatorial libraries, neuronal cells which are sensitive tohedgehog-dependent protection, such as dopaminergic and GABAergicneurons, can be contacted with a hedgehog protein and a test agent ofinterest, with the assay scoring for anything from simple binding to thecell to trophic responses by the target cell in the presence and absenceof the test agent. As with the cell-free assays, agents which produce astatistically significant change in hedgehog activities (eitherinhibition or potentiation) can be identified.

In other emdodiments, the cell-based assay scores for agents whichdisrupt association of patched and smoothened proteins, e.g., in thecell surface membrane or liposomal preparation.

In addition to characterizing cells that naturally express the patchedprotein, cells which have been genetically engineered to ectopicallyexpress patched can be utilized for drug screening assays. As anexample, cells which either express low levels or lack expression of thepatched protein, e.g. Xenopus laevis oocytes, COS cells or yeast cells,can be genetically modified using standard techniques to ectopicallyexpress the patched protein. (see Marigo et al., supra).

The resulting recombinant cells, e.g., which express a functionalpatched receptor, can be utilized in receptor binding assays to identifyagonist or antagonists of hedgehog binding. Binding assays can beperformed using whole cells. Furthermore, the recombinant cells of thepresent invention can be engineered to include other heterologous genesencoding proteins involved in hedgehog-dependent signal pathways. Forexample, the gene products of one or more of smoothened, costal-2 and/orfused can be co-expressed with patched in the reagent cell, with assaysbeing sensitive to the functional reconstituion of the hedgehog signaltransduction cascade.

Alternatively, liposomal preparations using reconstituted patchedprotein can be utilized. Patched protein purified from detergentextracts from both authentic and recombinant origins can bereconstituted in artificial lipid vesicles (e.g. phosphatidylcholineliposomes) or in cell membrane-derived vesicles (see, for example, Bearet al. (1992) Cell 68:809–818; Newton et al. (1983) Biochemistry22:6110–6117; and Reber et al. (1987) J Biol Chem 262:11369–11374). Thelamellar structure and size of the resulting liposomes can becharacterized using electron microscopy. External orientation of thepatched protein in the reconstituted membranes can be demonstrated, forexample, by immunoelectron microscopy. The hedgehog protein bindingactivity of liposomes containing patched and liposomes without theprotein in the presence of candidate agents can be compared in order toidentify potential modulators of the hedgehog-patched interaction.

The hedgehog protein used in these cell-based assays can be provided asa purified source (natural or recombinant in origin), or in the form ofcells/tissue which express the protein and which are co-cultured withthe target cells. As in the cell-free assays, where simple binding(rather than induction) is the hedgehog activity scored for in theassay, the protein can be labelled by any of the above-mentionedtechniques, e.g., fluorescently, enzymatically or radioactively, ordetected by immunoassay.

In addition to binding studies, functional assays can be used toidentified modulators, i.e., agonists of hedgehog or patched activities.By detecting changes in intracellular signals, such as alterations insecond messengers or gene expression in patched-expressing cellscontacted with a test agent, candidate antagonists to patched signalingcan be identified (e.g., having a hedgehog-like activity).

A number of gene products have been implicated in patched-mediatedsignal transduction, including patched, the transcription factor cubitusinterruptus (ci), the serine/threonine kinase fused (fu) and the geneproducts of costal-2, smoothened and suppressor of fused.

The interaction of a hedgehog protein with patched sets in motion acascade involving the activation and inhibition of downstream effectors,the ultimate consequence of which is, in some instances, a detectablechange in the transcription or translation of a gene. Potentialtranscriptional targets of patched signaling are the patched gene itself(Hidalgo and Ingham, 1990 Development 110, 291–301; Marigo et al., 1996)and the vertebrate homologs of the drosophila cubitus interruptus gene,the GLI genes (Hui et al. (1994) Dev Biol 162:402–413). Patched geneexpression has been shown to be induced in cells of the limb bud and theneural plate that are responsive to Shh. (Marigo et al. (1996) PNAS, inpress; Marigo et al. (1996) Development 122:1225–1233). The GLI genesencode putative transcription factors having zinc finger DNA bindingdomains (Orenic et al. (1990) Genes & Dev 4:1053–1067; Kinzler et al.(1990) Mol Cell Biol 10:634–642). Transcription of the GLI gene has beenreported to be upregulated in response to hedgehog in limb buds, whiletranscription of the GLI3 gene is downregulated in response to hedgehoginduction (Marigo et al. (1996) Development 122:1225–1233). By selectingtranscriptional regulatory sequences from such target genes, e.g. frompatched or GLI genes, that are responsible for the up- or downregulation of these genes in response to patched signalling, andoperatively linking such promoters to a reporter gene, one can derive atranscription based assay which is sensitive to the ability of aspecific test compound to modify patched signalling pathways. Expressionof the reporter gene, thus, provides a valuable screening tool for thedevelopment of compounds that act as antagonists of ptc, e.g., which maybe useful as neuroprotective agents.

Reporter gene based assays of this invention measure the end stage ofthe above described cascade of events, e.g., transcriptional modulation.Accordingly, in practicing one embodiment of the assay, a reporter geneconstruct is inserted into the reagent cell in order to generate adetection signal dependent on ptc signaling. To identify potentialregulatory elements responsive to ptc signaling present in thetranscriptional regulatory sequence of a target gene, nested deletionsof genomic clones of the target gene can be constructed using standardtechniques. See, for example, Current Protocols in Molecular Biology,Ausubel, F. M. et al. (eds.) Greene Publishing Associates, (1989); U.S.Pat. No. 5,266,488; Sato et al. (1995) J Biol Chem 270:10314–10322; andKube et al. (1995) Cytokine 7:1–7. A nested set of DNA fragments fromthe gene's 5′-flanking region are placed upstream of a reporter gene,such as the luciferase gene, and assayed for their ability to directreporter gene expression in patched expressing cells. Host cellstransiently transfected with reporter gene constructs can be scored forthe induction of expression of the reporter gene in the presence andabsence of hedgehog to determine regulatory sequences which areresponsie to patched-dependent signalling.

In practicing one embodiment of the assay, a reporter gene construct isinserted into the reagent cell in order to generate a detection signaldependent on second messengers generated by induction with hedgehogprotein. Typically, the reporter gene construct will include a reportergene in operative linkage with one or more transcriptional regulatoryelements responsive to the hedgehog activity, with the level ofexpression of the reporter gene providing the hedgehog-dependentdetection signal. The amount of transcription from the reporter gene maybe measured using any method known to those of skill in the art to besuitable. For example, mRNA expression from the reporter gene may bedetected using RNAse protection or RNA-based PCR, or the protein productof the reporter gene may be identified by a characteristic stain or anintrinsic activity. The amount of expression from the reporter gene isthen compared to the amount of expression in either the same cell in theabsence of the test compound (or hedgehog) or it may be compared withthe amount of transcription in a substantially identical cell that lacksthe target receptor protein. Any statistically or otherwise significantdifference in the amount of transcription indicates that the testcompound has in some manner altered the signal transduction of thepatched protein, e.g., the test compound is a potential ptc therapeutic.

As described in further detail below, in preferred embodiments the geneproduct of the reporter is detected by an intrinsic activity associatedwith that product. For instance, the reporter gene may encode a geneproduct that, by enzymatic activity, gives rise to a detection signalbased on color, fluorescence, or luminescence. In other preferredembodiments, the reporter or marker gene provides a selective growthadvantage, e.g., the reporter gene may enhance cell viability, relieve acell nutritional requirement, and/or provide resistance to a drug.

Preferred reporter genes are those that are readily detectable. Thereporter gene may also be included in the construct in the form of afusion gene with a gene that includes desired transcriptional regulatorysequences or exhibits other desirable properties. Examples of reportergenes include, but are not limited to CAT (chloramphenicol acetyltransferase) (Alton and Vapnek (1979), Nature 282: 864–869) luciferase,and other enzyme detection systems, such as beta-galactosidase; fireflyluciferase (deWet et al. (1987), Mol. Cell. Biol. 7:725–737); bacterialluciferase (Engebrecht and Silverman (1984), PNAS 1: 4154–4158; Baldwinet al. (1984), Biochemistry 23: 3663–3667); alkaline phosphatase (Toh etal. (1989) Eur. J. Biochem. 182: 231–238, Hall et al. (1983) J. Mol.Appl. Gen. 2: 101), human placental secreted alkaline phosphatase(Cullen and Malim (1992) Methods in Enzymol. 216:362–368).

Transcriptional control elements which may be included in a reportergene construct include, but are not limited to, promoters, enhancers,and repressor and activator binding sites. Suitable transcriptionalregulatory elements may be derived from the transcriptional regulatoryregions of genes whose expression is induced after modulation of apatched signal transduction pathway. The characteristics of preferredgenes from which the transcriptional control elements are derivedinclude, but are not limited to, low or undetectable expression inquiescent cells, rapid induction at the transcriptional level withinminutes of extracellular stimulation, induction that is transient andindependent of new protein synthesis, subsequent shut-off oftranscription requires new protein synthesis, and mRNAs transcribed fromthese genes have a short half-life. It is not necessary for all of theseproperties to be present.

In yet other embodiments, second messenger generation can be measureddirectly in the detection step, such as mobilization of intracellularcalcium, phospholipid metabolism or adenylate cyclase activity arequantitated, for instance, the products of phospholipid hydrolysis IP₃,DAG or cAMP could be measured For example, recent studies haveimplicated protein kinase A (PKA) as a possible component ofhedgehog/patched signaling (Hammerschmidt et al. (1996) Genes & Dev10:647). High PKA activity has been shown to antagonize hedgehogsignaling in these systems. Conversely, inhibitors of PKA will mimicand/or potentiate the action of hedgehog. Although it is unclear whetherPKA acts directly downstream or in parallel with hedgehog signaling, itis possible that hedgehog signalling occurs via inhibition of PKAactivity. Thus, detection of PKA activity provides a potential readoutfor the instant assays. In certain embodiments, a preferred ptctherapeutic inhibits PKA with a K_(i) less than 10 nM, preferably lessthan 1 nM, even more preferably less than 0.1 nM.

In a preferred embodiment, the ptc therapeutic is a PKA inhibitor. Avariety of PKA inhibitors are known in the art, including both peptidyland organic compounds. For instance, the ptc therapeutic can be a5-isoquinolinesulfonamide, such as represented in the general formula:

wherein,

R₁ and R₂ each can independently represent hydrogen, and as valence andstability permit a lower alkyl, a lower alkenyl, a lower alkynyl, acarbonyl (such as a carboxyl, an ester, a formate, or a ketone), athiocarbonyl (such as a thioester, a thioacetate, or a thioformate), anamino, an acylamino, an amido, a cyano, a nitro, an azido, a sulfate, asulfonate, a sulfonamido, —(CH₂)_(m)—R₈, —(CH₂)_(m)—OH,—(CH₂)_(m)—O-lower alkyl, —(CH₂)_(m)—O-lower alkenyl,—(CH₂)_(n)—O—(CH₂)_(m)—R₈, —(CH₂)_(m)—SH, —(CH₂)_(m)—S-lower alkyl,—(CH₂)_(m)—S-lower alkenyl, —(CH₂)_(n)—S—(CH₂)_(m)—R₈, or

R₁ and R₂ taken together with N form a heterocycle (substituted orunsubstituted);

R₃ is absent or represents one or more substitutions to the isoquinolinering such as a lower alkyl, a lower alkenyl, a lower alkynyl, a carbonyl(such as a carboxyl, an ester, a formate, or a ketone), a thiocarbonyl(such as a thioester, a thioacetate, or a thioformate), an amino, anacylamino, an amido, a cyano, a nitro, an azido, a sulfate, a sulfonate,a sulfonamido, —(CH₂)_(m)—R₈, —(CH₂)_(m)—OH, —(CH₂)_(m)—O-lower alkyl,—(CH₂)_(m)—O-lower alkenyl, —(CH₂)_(n)—O—(CH₂)_(m)—R₈, —(CH₂)_(m)—SH,—(CH₂)_(m)—S-lower alkyl, —(CH₂)_(m)—S-lower alkenyl,—(CH₂)_(n)—S—(CH₂)_(m)—R₈;

R₈ represents a substituted or unsubstituted aryl, aralkyl, cycloalkyl,cycloalkenyl, or heterocycle; and

n and m are independently for each occurrence zero or an integer in therange of 1 to 6. In a preferred embodiment, the PKA inhibitor isN-[2-((p-bromocinnamyl)amino)ethyl]-5-isoquinolinesulfonamide (H-89;Calbiochem Cat. No. 371963), e.g., having the formula:

In another embodiment, the PKA inhibitor is1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H-7; Calbiochem Cat. No.371955), e.g., having the formula:

In still other embodiments, the PKA inhibitor is KT5720 (Calbiochem Cat.No. 420315), having the structure

The hedgehog pathway can be agonized by antagonizing the cAMP pathway,e.g., by using an agonist of of cAMP phosphodiesterase, or by using anantagonist of adenylate cyclase, cAMP or protein kinase A (PKA).Compounds which may reduce the levels or activity of cAMP includeprostaglandylinositol cyclic phosphate (cyclic PIP), endothelins (ET)-1and -3, norepinepurine, K252a, dideoxyadenosine, dynorphins, melatonin,pertussis toxin, staurosporine, G₁ agonists, MDL 12330A, SQ 22536,GDPssS and clonidine, beta-blockers, and ligands of G-protein coupledreceptors. Additional compounds are disclosed in U.S. Pat. Nos.5,891,875, 5,260,210, and 5,795,756.

Exemplary peptidyl inhibitors of PKA activity include the PKA HeatStable Inhibitor (isoform α; see, for example, Calbiochem Cat. No.539488, and Wen et al. (1995) J Biol Chem 270:2041).

In certain embodiments, a compound which is an agonist or antagonist ofPKA is chosen to be selective for PKA over other protein kinases, suchas PKC, e.g., the compound modulates the activity of PKA at least anorder of magnitude more strongly than it modulates the activity ofanother protein kinase, preferably at least two orders of magnitude morestrongly, even more preferably at least three orders of magnitude morestrongly. Thus, for example, a preferred inhibitor of PKA may inhibitPKA activity with a K_(i) at least an order of magnitude lower than itsK_(i) for inhibition of PKC, preferably at least two orders of magnitudelower, even more preferably at least three orders of magnitude lower. Incertain embodiments, a ptc therapeutic inhibits PKC with a K_(i) greaterthan 10 nM, greater than 100 nM, preferably greater than 1 μM.

Certain hedgehog receptors may stimulate the activity of phospholipases.Inositol lipids can be extracted and analyzed using standard lipidextraction techniques. Water soluble derivatives of all three inositollipids (IP₁, IP₂, IP₃) can also be quantitated using radiolabellingtechniques or HPLC.

The mobilization of intracellular calcium or the influx of calcium fromoutside the cell may be a response to hedgehog stimulation or lack thereof. Calcium flux in the reagent cell can be measured using standardtechniques. The choice of the appropriate calcium indicator,fluorescent, bioluminescent, metallochromic, or Ca⁺⁺-sensitivemicroelectrodes depends on the cell type and the magnitude and timeconstant of the event under study (Borle (1990) Environ Health Perspect84:45–56). As an exemplary method of Ca⁺⁺ detection, cells could beloaded with the Ca⁺⁺ sensitive fluorescent dye fura-2 or indo-1, usingstandard methods, and any change in Ca⁺⁺ measured using a fluorometer.

In certain embodiments of the assay, it may be desirable to screen forchanges in cellular phosphorylation. As an example, the drosophila genefused (fu) which encodes a serine/threonine kinase has been identifiedas a potential downstream target in hedgehog signaling. (Preat et al.,1990 Nature 347, 87–89; Therond et al. 1993, Mech. Dev. 44. 65–80). Theability of compounds to modulate serine/threonine kinase activationcould be screened using colony immunoblotting (Lyons and Nelson (1984)Proc. Natl. Acad. Sci. USA 81:7426–7430) using antibodies againstphosphorylated serine or threonine residues. Reagents for performingsuch assays are commercially available, for example, phosphoserine andphosphothreonine specific antibodies which measure increases inphosphorylation of those residues can be purchased from commercialsources.

In yet another embodiment, the ptc therapeutic is an antisense moleculewhich inhibits expression of a protein involved in a patched-mediatedsignal transduction pathway. To illustrate, by inhibiting the expressionof a protein involved in patched signals, such as fused, costal-2,smoothened and/or Gli genes, or patched itself, the ability of thepatched signal pathway(s) to alter the ability of, e.g., a dopaminergicor GABAergic cell to maintain its differentiated state can be altered,e.g., potentiated or repressed.

As used herein, “antisense” therapy refers to administration or in situgeneration of oligonucleotide probes or their derivatives whichspecifically hybridize (e.g. bind) under cellular conditions withcellular mRNA and/or genomic DNA encoding a hedgehog protein, patched,or a protein involved in patched-mediated signal transduction. Thehybridization should inhibit expression of that protein, e.g. byinhibiting transcription and/or translation. The binding may be byconventional base pair complementarity, or, for example, in the case ofbinding to DNA duplexes, through specific interactions in the majorgroove of the double helix. In general, “antisense” therapy refers tothe range of techniques generally employed in the art, and includes anytherapy which relies on specific binding to oligonucleotide sequences.

An antisense construct of the present invention can be delivered, forexample, as an expression plasmid which, when transcribed in the cell,produces RNA which is complementary to at least a unique portion of thetarget cellular mRNA. Alternatively, the antisense construct is anoligonucleotide probe which is generated ex vivo and which, whenintroduced into the cell causes inhibition of expression by hybridizingwith the mRNA and/or genomic sequences of a target gene. Sucholigonucleotide probes are preferably modified oligonucleotide which areresistant to endogenous nucleases, e.g. exonucleases and/orendonucleases, and is therefore stable in vivo. Exemplary nucleic acidmolecules for use as antisense oligonucleotides are phosphoramidate,phosphothioate and methylphosphonate analogs of DNA (see also U.S. Pat.Nos. 5,176,996; 5,264,564; and 5,256,775). Additionally, generalapproaches to constructing oligomers useful in antisense therapy havebeen reviewed, for example, by Van der Krol et al. (1988) Biotechniques6:958–976; and Stein et al. (1988) Cancer Res 48:2659–2668.

Several considerations should be taken into account when constructingantisense oligonucleotides for the use in the methods of the invention:(1) oligos should have a GC content of 50% or more; (2) avoid sequenceswith stretches of 3 or more G's; and (3) oligonucleotides should not belonger than 25–26 mers. When testing an antisense oligonucleotide, amismatched control can be constructed. The controls can be generated byreversing the sequence order of the corresponding antisenseoligonucleotide in order to conserve the same ratio of bases.

5′-GTCCTGGCGCCGCCGCCGCCGTCGCC (SEQ ID NO: 26)5′-TTCCGATGACCGGCCTTTCGCGGTGA (SEQ ID NO: 27)5′-GTGCACGGAAAGGTGCAGGCCACACT (SEQ ID NO: 28)VI. Exemplary Pharmaceutical Preparations of Hedgehog and ptcTherapeutics

The source of the hedgehog and ptc therapeutics to be formulated willdepend on the particular form of the agent. Small organic molecules andpeptidyl fragments can be chemically synthesized and provided in a pureform suitable for pharmaceutical/cosmetic usage. Products of naturalextracts can be purified according to techniques known in the art. Forexample, the Cox et al. U.S. Pat. No. 5,286,654 describes a method forpurifying naturally occurring forms of a secreted protein and can beadapted for purification of hedgehog polypeptides. Recombinant sourcesof hedgehog polypeptides are also available. For example, the geneencoding hedgehog polypeptides, are known, inter alia, from PCTpublications WO 95/18856 and WO 96/17924.

Those of skill in treating neural tissues can determine the effectiveamount of an hedgehog or ptc therapeutic to be formulated in apharmaceutical or cosmetic preparation.

The hedgehog or ptc therapeutic formulations used in the method of theinvention are most preferably applied in the form of appropriatecompositions. As appropriate compositions there may be cited allcompositions usually employed for systemically or locally (such asintrathecal) administering drugs. The pharmaceutically acceptablecarrier should be substantially inert, so as not to act with the activecomponent. Suitable inert carriers include water, alcohol polyethyleneglycol, mineral oil or petroleum gel, propylene glycol and the like.

To prepare the pharmaceutical compositions of this invention, aneffective amount of the particular hedgehog or ptc therapeutic as theactive ingredient is combined in intimate admixture with apharmaceutically acceptable carrier, which carrier may take a widevariety of forms depending on the form of preparation desired foradministration. These pharmaceutical compositions are desirable inunitary dosage form suitable, particularly, for administration orally,rectally, percutaneously, or by parenteral injection. For example, inpreparing the compositions in oral dosage form, any of the usualpharmaceutical media may be employed such as, for example, water,glycols, oils, alcohols and the like in the case of oral liquidpreparations such as suspensions, syrups, elixirs and solutions; orsolid carriers such as starches, sugars, kaolin, lubricants, binders,disintegrating agents and the like in the case of powders, pills,capsules, and tablets. Because of their ease in administration, tabletsand capsules represents the most advantageous oral dosage unit form, inwhich case solid pharmaceutical carriers are obviously employed. Forparenteral compositions, the carrier will usually comprise sterilewater, at least in large part, though other ingredients, for example, toaid solubility, may be included. Injectable solutions, for example, maybe prepared in which the carrier comprises saline solution, glucosesolution or a mixture of saline and glucose solution. Injectablesuspensions may also be prepared in which case appropriate liquidcarriers, suspending agents and the like may be employed. Also includedare solid form preparations which are intended to be converted, shortlybefore use, to liquid form preparations. In the compositons suitable forpercutaneous administration, the carrier optionally comprises apenetration enhancing agent and/or a suitable wetting agent, optionallycombined with suitable additives of any nature in minor proportions,which additives do not introduce a significant deleterious effect on theskin.

It is especially advantageous to formulate the subject compositions indosage unit form for ease of administration and uniformity of dosage.Dosage unit form as used in the specification and claims herein refersto physically discrete units suitable as unitary dosages, each unitcontaining a predetermined quantity of active ingredient calculated toproduce the desired therapeutic effect in association with the requiredpharmaceutical carrier. Examples of such dosage unit forms are tablets(including scored or coated tablets), capsules, pills, powders packets,wafers, injectable solutions or suspensions, teaspoonfuls,tablespoonfuls and the like, and segregated multiples thereof.

The pharmaceutical preparations of the present invention can be used, asstated above, for the many applications which can be considered cosmeticuses. Cosmetic compositions known in the art, preferably hypoallergicand pH controlled are especially preferred, and include toilet waters,packs, lotions, skin milks or milky lotions. The preparations contain,besides the hedgehog or ptc therapeutic, components usually employed insuch preparations. Examples of such components are oils, fats, waxes,surfactants, humectants, thickening agents, antioxidants, viscositystabilizers, chelating agents, buffers, preservatives, perfumes,dyestuffs, lower alkanols, and the like. If desired, further ingredientsmay be incorporated in the compositions, e.g. antiinflammatory agents,antibacterials, antifungals, disinfectants, vitamins, sunscreens,antibiotics, or other anti-acne agents.

Examples of oils comprise fats and oils such as olive oil andhydrogenated oils; waxes such as beeswax and lanolin; hydrocarbons suchas liquid paraffin, ceresin, and squalane; fatty acids such as stearicacid and oleic acid; alcohols such as cetyl alcohol, stearyl alcohol,lanolin alcohol, and hexadecanol; and esters such as isopropylmyristate, isopropyl palmitate and butyl stearate. As examples ofsurfactants there may be cited anionic surfactants such as sodiumstearate, sodium cetylsulfate, polyoxyethylene laurylether phosphate,sodium N-acyl glutamate; cationic surfactants such asstearyldimethylbenzylammonium chloride and stearyltrimethylammoniumchloride; ampholytic surfactants such as alkylaminoethylglycinehydrocloride solutions and lecithin; and nonionic surfactants such asglycerin monostearate, sorbitan monostearate, sucrose fatty acid esters,propylene glycol monostearate, polyoxyethylene oleylether, polyethyleneglycol monostearate, polyoxyethylene sorbitan monopalmitate,polyoxyethylene coconut fatty acid monoethanolamide, polyoxypropyleneglycol (e.g. the materials sold under the trademark “Pluronic”),polyoxyethylene castor oil, and polyoxyethylene lanolin. Examples ofhumectants include glycerin, 1,3-butylene glycol, and propylene glycol;examples of lower alcohols include ethanol and isopropanol; examples ofthickening agents include xanthan gum, hydroxypropyl cellulose,hydroxypropyl methyl cellulose, polyethylene glycol and sodiumcarboxymethyl cellulose; examples of antioxidants comprise butylatedhydroxytoluene, butylated hydroxyanisole, propyl gallate, citric acidand ethoxyquin; examples of chelating agents include disodium edetateand ethanehydroxy diphosphate; examples of buffers comprise citric acid,sodium citrate, boric acid, borax, and disodium hydrogen phosphate; andexamples of preservatives are methyl parahydroxybenzoate, ethylparahydroxybenzoate, dehydroacetic acid, salicylic acid and benzoicacid.

For preparing ointments, creams, toilet waters, skin milks, and thelike, typically from 0.01 to 10% in particular from 0.1 to 5% and morein particular from 0.2 to 2.5% of the active ingredient, e.g., of thehedgehog or ptc therapeutic, will be incorporated in the compositions.In ointments or creams, the carrier for example consists of 1 to 20%, inparticular 5 to 15% of a humectant, 0.1 to 10% in particular from 0.5 to5% of a thickener and water; or said carrier may consist of 70 to 99%,in particular 20 to 95% of a surfactant, and 0 to 20%, in particular 2.5to 15% of a fat; or 80 to 99.9% in particular 90 to 99% of a thickener;or 5 to 15% of a surfactant, 2–15% of a humectant, 0 to 80% of an oil,very small (<2%) amounts of preservative, coloring agent and/or perfume,and water. In a toilet water, the carrier for example consists of 2 to10% of a lower alcohol, 0.1 to 10% or in particular 0.5 to 1% of asurfactant, 1 to 20%, in particular 3 to 7% of a humectant, 0 to 5% of abuffer, water and small amounts (<2%) of preservative, dyestuff and/orperfume. In a skin milk, the carrier typically consists of 10–50% ofoil, 1 to 10% of surfactant, 50–80% of water and 0 to 3% of preservativeand/or perfume. In the aforementioned preparations, all % symbols referto weight by weight percentage.

Particular compositions for use in the method of the present inventionare those wherein the hedgehog or ptc therapeutic is formulated inliposome-containing compositions. Liposomes are artificial vesiclesformed by amphiphatic molecules such as polar lipids, for example,phosphatidyl cholines, ethanolamines and serines, sphingomyelins,cardiolipins, plasmalogens, phosphatidic acids and cerebiosides.Liposomes are formed when suitable amphiphathic molecules are allowed toswell in water or aqueous solutions to form liquid crystals usually ofmultilayer structure comprised of many bilayers separated from eachother by aqueous material (also referred to as coarse liposomes).Another type of liposome known to be consisting of a single bilayerencapsulating aqueous material is referred to as a unilamellar vesicle.If water-soluble materials are included in the aqueous phase during theswelling of the lipids they become entrapped in the aqueous layerbetween the lipid bilayers.

Water-soluble active ingredients such as, for example, various saltforms of a hedgehog polypeptide, are encapsulated in the aqueous spacesbetween the molecular layers. The lipid soluble active ingredient ofhedgehog or ptc therapeutic, such as an organic mimetic, ispredominantly incorporated into the lipid layers, although polar headgroups may protude from the layer into the aqueous space. Theencapsulation of these compounds can be achieved by a number of methods.The method most commonly used involves casting a thin film ofphospholipid onto the walls of a flask by evaporation from an organicsolvent. When this film is dispersed in a suitable aqueous medium,multilamellar liposomes are formed. Upon suitable sonication, the coarseliposomes form smaller similarly closed vesicles.

Water-soluble active ingredients are usually incorporated by dispersingthe cast film with an aqueous solution of the compound. Theunencapsulated compound is then removed by centrifugation,chromatography, dialysis or other art-known suitable procedures. Thelipid-soluble active ingredient is usually incorporated by dissolving itin the organic solvent with the phospholipid prior to casting the film.If the solubility of the material in the lipid phase is not exceeded orthe amount present is not in excess of that which can be bound to thelipid, liposomes prepared by the above method usually contain most ofthe material bound in the lipid bilayers; separation of the liposomesfrom unencapsulated material is not required.

A particularly convenient method for preparing liposome formulated formsof hedgehog and ptc therapeutics is the method described inEP-A-253,619, incorporated herein by reference. In this method, singlebilayered liposomes containing encapsulated active ingredients areprepared by dissolving the lipid component in an organic medium,injecting the organic solution of the lipid component under pressureinto an aqueous component while simultaneously mixing the organic andaqueous components with a high speed homogenizer or mixing means,whereupon the liposomes are formed spontaneously.

The single bilayered liposomes containing the encapsulated hedgehog orptc therapeutic can be employed directly or they can be employed in asuitable pharmaceutically acceptable carrier for localizedadministration. The viscosity of the liposomes can be increased by theaddition of one or more suitable thickening agents such as, for examplexanthan gum, hydroxypropyl cellulose, hydroxypropyl methylcellulose andmixtures thereof. The aqueous component may consist of water alone or itmay contain electrolytes, buffered systems and other ingredients, suchas, for example, preservatives. Suitable electrolytes which can beemployed include metal salts such as alkali metal and alkaline earthmetal salts. The preferred metal salts are calcium chloride, sodiumchloride and potassium chloride. The concentration of the electrolytemay vary from zero to 260 mM, preferably from 5 mM to 160 mM. Theaqueous component is placed in a suitable vessel which can be adapted toeffect homogenization by effecting great turbulence during the injectionof the organic component. Homogenization of the two components can beaccomplished within the vessel, or, alternatively, the aqueous andorganic components may be injected separately into a mixing means whichis located outside the vessel. In the latter case, the liposomes areformed in the mixing means and then transferred to another vessel forcollection purpose.

The organic component consists of a suitable non-toxic, pharmaceuticallyacceptable solvent such as, for example ethanol, glycerol, propyleneglycol and polyethylene glycol, and a suitable phospholipid which issoluble in the solvent. Suitable phospholipids which can be employedinclude lecithin, phosphatidylcholine, phosphatydylserine,phosphatidylethanolamine, phosphatidylinositol, lysophosphatidylcholineand phospha-tidyl glycerol, for example. Other lipophilic additives maybe employed in order to selectively modify the characteristics of theliposomes. Examples of such other additives include stearylamine,phosphatidic acid, tocopherol, cholesterol and lanolin extracts.

In addition, other ingredients which can prevent oxidation of thephospholipids may be added to the organic component. Examples of suchother ingredients include tocopherol, butylated hydroxyanisole,butylated hydroxytoluene, ascorbyl palmitate and ascorbyl oleate.Preservatives such a benzoic acid, methyl paraben and propyl paraben mayalso be added.

Methods of introduction may also be provided by rechargeable orbiodegradable devices. Various slow release polymeric devices have beendeveloped and tested in vivo in recent years for the controlled deliveryof drugs, including proteinacious biopharmaceuticals. A variety ofbiocompatible polymers (including hydrogels), including bothbiodegradable and non-degradable polymers, can be used to form animplant for the sustained release of an hh at a particular target site.Such embodiments of the present invention can be used for the deliveryof an exogenously purified hedgehog protein, which has been incorporatedin the polymeric device, or for the delivery of hedgehog produced by acell encapsulated in the polymeric device.

An essential feature of certain embodiments of the implant can be thelinear release of the therapeutic, which can be achieved through themanipulation of the polymer composition and form. By choice of monomercomposition or polymerization technique, the amount of water, porosityand consequent permeability characteristics can be controlled. Theselection of the shape, size, polymer, and method for implantation canbe determined on an individual basis according to the disorder to betreated and the individual patient response. The generation of suchimplants is generally known in the art. See, for example, ConciseEncylopedia of Medical & Dental Materials, ed. by David Williams (MITPress: Cambridge, Mass., 1990); and the Sabel et al. U.S. Pat. No.4,883,666.

In another embodiment of an implant, a source of cells producing thetherapeutic, e.g., secreting a soluble form of a hedgehog protein, isencapsulated in implantable hollow fibers or the like. Such fibers canbe pre-spun and subsequently loaded with the cell source (Aebischer etal. U.S. Pat. No. 4,892,538; Aebischer et al. U.S. Pat. No. 5,106,627;Hoffman et al. (1990) Expt. Neurobiol. 110:39–44; Jaeger et al. (1990)Prog. Brain Res. 82:41–46; and Aebischer et al. (1991) J. Biomech. Eng.113:178–183), or can be co-extruded with a polymer which acts to form apolymeric coat about the cells (Lim U.S. Pat. No. 4,391,909; Sefton U.S.Pat. No. 4,353,888; Sugamori et al. (1989) Trans. Am. Artif. Intern.Organs 35:791–799; Sefton et al. (1987) Biotehnol. Bioeng. 29:1135–1143;and Aebischer et al. (1991) Biomaterials 12:50–55).

EXEMPLIFICATION

The invention now being generally described, it will be more readilyunderstood by reference to the following examples which are includedmerely for purposes of illustration of certain aspects and embodimentsof the present invention, which are not intended to limit the invention.

In Drosophila, the hedgehog gene was first discovered for the role itplays in early embryo patterning (Nusslein-Volhard and Wieschaus, 1980).Further study showed tht the product of this gene is secreted, and as anintercellular signaling protein, plays a critical role in bodysegmentation and patterning of imaginal disc derivatives such as eyesand wings (Lee et al., 1992; Mohler and Vanie, 1992; Tabata et al.,1992). There re, at present, three mammalian homologues of Drosophilahedgehog, and Indian hedgehog (Fietz et al., 1994). During the course ofvertebrate development, these secreted peptide molecules are involved inaxial patterning, and consequently regulate the phenotypic specificationof precursor cells into functional differentiated cells.

The embryonic expression pattern of Shh has been shown to be closelylinked to the development and differentiation of the entire ventralneuraxis (Marti et al., 1995). Using naive neural tube explants derivedfrom the appropriate levels of the rostrocaudal axis, it has beendemonstrated that the induction of spinal motor neurons (Roelink et al.,1994; Tanabe et al., 1995), midbrain dopaminergic neurons (Hynes et al.,1995; Wang et al., 1995), and basal forebrain cholinergic neurons(Ericson et al., 1995) are dependent upon exposure to Shh. This moleculeappears to be crucial for such patterning and phenotype specification invivo since mouse embryos deficient in the expression of functional Shhgene product manifest a lack of normal ventral patterning in the centralnervous system as well as gross atrophy of the entire cranium (Chiang etal., 1996).

In this study we have explored the issue of whether Shh may haveactivities at stages in neural development later than those previouslystudied. Namely, we have asked whether Shh is trophic for particularneural populations, and under toxic conditions, whethre Shh isneuroprotective. Using cultures derived from the embryonic day 14–16(E14–16) rat, we find that Shh is trophic for midbrain, striatial, andspinal neurons. In the first case the factor is trophic for bothdopaminergic and GABA-immunoreactive (GABA-ir) neurons. From thestriatum, the surviving neurons are exclusively GABA-ir, while in thespinal cultures Shh promotes survival of a heterogeneous population ofputative interneurons. Shh does not suport survival of any peripheralnervous system neurons tested. Finally, we show that Shh protectscultures of midbrain dopaminergic neurons from the toxic effects ofMPP+, a specific neurotoxin that induces Parkinsonism in vivo. Together,these observations indicate a novel role for Shh in nervous systemdevelopment and its potential role as a therapeutic.

MATERIALS AND METHODS

Whole-Mount in situ Hybridization

Whole-mount in situ hybridization on bisected E14.5 Sprague-Dawley ratembryos was performed with digoxigenin-labeled (Boehringer-Mannheim)mouse RNA probes as previously described (Wilkinson, 1992). Bound probewas detected with alkaline phosphatase-conjugated anti-digoxigenin Fabfragments (BoehringerMannheim). The 0.7 kb Shh probes were transcribedusing T3 (antisense) or T7 (sense) RNA polymerase from Hind III(antisense) or Bam HI (sense) linearized templates as described byEchelard, et al. (1993). The 0.9 kb Ptc probes were transcribed using T3(antisense) or T7 (sense) RNA polymerase from Bam HI (antisense) or HindIII (sense) linearized templates as described by Goodrich, et al.(1996).

Shh Protein and Anti-Shh Antibody

Rat sonic hedgehog amino terminal signaling domain (amino acids 2–198)Porter et al., 1995) was cloned into a baculovirus expression vector(Invitrogen; San Diego, Calif.) (virus encoding Shh insert was a gift ofDr. Henk Roelink, University of Washington, Seattle, Wash.). High Five™insect cells (Invitrogen) were infected with the baculovirus permanufacturer's instructions. The culture supernatant was batch adsorbedto heparin agarose type I (Sigma; St. Louis, Mo.) and Shh eluted withPBS containing a total of 0.75 M NaCl and 0.1 mM mercaptoethanol. Shhconcentration was determined by the method of Ericson, et al. (1996). E.coli-derived Shh was obtained as previously described (Wang et al.,1996) and purified as described above. All samples were sterile filteredand aliquots frozen in liquid nitrogen. Anti-Shh polyclonal antibody wasa gift from Dr. Andy McMahon (Harvard University). Preparation of thisreagent, directed against the amino peptide of Shh, is described byBumcrot et al. (1995). Anti-Shh monoclonal antibody (511) was a gift ofDr. Thomas Jessell (Columbia University), and preparation of thisreagent is described by Ericson et al. (1996).

Dissociation and Culture of Neural Tissue

E14.5 rat ventral mesencephalon was dissected as described by Shimoda,et al. (Shimoda et al., 1992). Striatal cultures were established fromE15–16 embryos from the regions identified by Altman and Bayer (1995) asthe striatum and pallidum. Spinal cultures utilized the ventralone-third of the E15–16 spinal cord (Camu and Henderson, 1992). Tissueswere dissociated for approximately 40 minutes in 0.10–0.25% trypsin-EDTA(Gibco/BRL; Gaithersburg, Md.), and the digestion stopped using an equalvolume of Ca++/Mg++-free Hanks' buffered saline (Gibco/BRL) containing3.5 mg/ml soybean trypsin inhibitor (Sigma) and 0.04% DNase (Grade II,Boehringer Mannheim; Indianapolis, Ind.). Cells were than plated at2×20⁵−3×20⁵ cells/well in the medium of Krieglstein, et al. (1995) (amodified N2 medium) in 34-well tissue culture plates (Falcon) coatedwith poly-L-lysine or poly-L-ornithine (Sigma) after 2 wash in the samemedium. Note that this procedure results in cultures in which the cellshave never been exposed to serum and stands in contrast to cultures inwhich serum has been used to neutralize dissociation proteases, and/orto intially “prime” the cells prior to serum withdrawal. The followingpeptide growth factors were added as indicated in the results: basicfibroblast growth factor (FGFb), transforming growth factor 1(TGF 1),TGF 2, glia-derived neurotrophic factor (GDNF), and brain derivedneurotrophic factor (BDNF) (all from PeproTech; Rocky Hill, N.J.;additional lots of BDNF and GDNF were purchased from Promega; MadisonWis.). Anti-TGF antibodies were purchased from R & D Systems. Antibodywas added at the time of Shh addition to the cultures. Cultures weremaintained for up to 3 weeks and the medium changed every 4 days.

Immunocytyochemistry and Cell Scoring

For all cell staining, cultures were fixed with 5% paraformaldehyde inPBS (plus 0.1% glutaraldehyde if staining for GABA), and blocked using3% goat serum, (Sigma), 0.1% Triton X-100, in PBS. Antibody incubationswere performed in the blocking solutions. Antibodies used in this studywere anti-tubulin III (Sigma), anti-tyrosine hydroxylase (TH)(Boehringer-Mannheim), anti-GABA (Sigma), and anti-glial fibrillaryacidic protein (GFAP) (Sigma). Primary antibodies were detected usinghorseradish peroxidase-, alkaline phosphatase-, orflurochrome-conjugated secondary antibodies (Vector; Burlingame,Calif.). Peroxidase-linked secondaries were visualized using a NI/DABkit (Zymed; South San Francisco, Calif.) and phosphatase-linkedsecondaries using Vector Blue™ (Vector).

Cell counting was performed using an Olympus inverted microscope at atotal magnification of 300×. Data presented are representative, and havebeen confirmed by repeating the cultures at least 4–10 independent timesfor each neural population discussed. Cell numbers are reported ascells/field (the average of 30–40 fields from a total of 5wells/condition; 4–10 independent experiments were assessed for eachculture condtion examined). Consistency of counting was verified by atleast 3 observers. Errors are reported as standard error of the mean(s.e.m.), and significance calculated by student's t-test.

Measurement of Dopamine Transport

To detect the presence of the dopamine transporter (Cerruti et al.,1993; Ciliax et al., 1995) cultures were incubated with a mixtureconsisting of: 5×10⁻⁸M ³H-dopamine (Amersham; Arlington Heights, Ill.;48 Ci/mmol), 100 μM ascorbic acid (Sigma), 1 μM fluoxetine (Eli Lilly;Indianapolis, Ind.), 1 μM desmethylimipramine (Sigma), and 10 μMpargyline (Sigma) in DME-F12. Nonspecific labeling was measured by theaddition of 5×10⁻⁵M unlabeled dopamine. Cells were incubated for 30minutes at 37° C., rinsed three times with PBS and processed for eitherscintillation counting or autoradiography. For scintillation counting,cells were first lysed with 150 μl of 0.1% SDS and then added to 500 μlof Microscint 20 (Packard; Meriden, Conn.) and counted in a PackardInstrument Topcount scintillation machine. For autoradiography, sisterplates were coated with NTB-2 autoradiographic emulsion (Kodak;Rochester, N.Y.) that had been diluted 1:3 with 10% glycerol. The plateswere then air dried, exposed for 1–2 weeks, and developed.

Quantitative-Competitive Polymerase Chain Reaction (QC-PCR)

RNA was isolated from cells and tissue using Trizol (Gibco/BRL) asprescribed by the manufacturer. Genomic DNA was removed from the RNA byincubation with 0.5 units of Dnase (Gibco/BRL, Cat # 28068-015) at roomtemperature for 25 minutes. The solution was heated to 75 C for 20minutes to inactivate the DNase. Reverse transcription was carried outusing random hexamer and MuLV reverse transcriptase (Gibco/BRL) assuggested by the manufacturer. All the quantitative RT-PCR internalcontrols, or mimics, were synthetic single stranded DNA oligonucleotidescorresponding to the target sequence with an internal deletion from thecentral region (Oligos, Etc.; Wilsonville, Oreg.). For actin, target=280bp, mimic=230 bp; for ptc, target=354 bp, mimic=200 bp. PCR wasperformed using the Clontech RCR kit. For actin: annealing temperature64° C., oligos GGCTCCGGTATGTGC (SEQ ID NO: 29), GGGGTACTTCAGGGT (SEQ IDNO: 30). For ptc: annealing temperature 72° C., oligosCATTGGCAGGAGGAGTTGATTGTGG (SEQ ID NO: 31), AGCACCTTTTGAGTGGAGTTTGGGG(SEQ ID NO: 32). In each QC-PCR reaction, four reactions were set upwith equal amounts of sample cDNA in each tube and 5-fold serialdilution of mimic. Also, for each sample an aliquot of cDNA was savedand amplified along with quantitative PCR as control for contamination.PCR reactions were carried out in an MJ Research PTC-200 thermal cyclerand the following cycling profile used: 95° C. for 45 seconds, 64 or 72°C. for 35 seconds, 82° C. for 30 seconds; for 40 cycles. The reactionmixtures were then fractionated by agarose electrophoresis, negativefilms obtained, and the films digitally scanned and quantified by areaintegration according to established procedures (Wang et al., 1995, andreferences therein). The quantity of target molecules was normalized tothe competing mimic and expressed as a function of cDNA synthesized andused in each reaction.

N-methyl-4-phenylpyrridinium (MPP+) Administration

Culture and MPP+ treatment of dopaminergic neurons were performed aspreviously described (Hyman et al., 1994; Krieglstein et al., 1995).MPP+ (Aldrich; St. Louis, Mo.) was added at day 3 of culture to a finalconcentration of 3 μM for 58 hours. Cultures were then washedextensively to remove MPP+, cultured for an additional 34–48 hours toallow clearance of dying TH+ neurons, and then processed forimmunocytochemistry.

RESULTS

Shh and Ptc Continue to be Expressed in the Rat CNS After the MajorPeriod of Dorsoventral Patterning

Previous studies have shown that shh is expressed in the vertebrateembryo in the period during which dorsoventral patterning manifests(approximately E9–10 in the rat). Within the central nervous system, shhexpression persists beyond this period and can be detected at a veryhigh level in the E14–16 rat embryo. For example, in situ hybridizationstudies of the E14.5 embryo (FIGS. 1A and E) reveal that shh isexpressed in ventral regions of the spinal cord, hindbrain, midbrain,and diencephalon. Lower levels of expression are observed in the ventralstriatum and septum, while no expression is observed in the cortexwithin the limits of detection of this method. Interestingly, a “streak”of shh expression (FIG. 1A, arrow) is observed to bisect thediencephalon into rostral and caudal halves. This is likely to be thezona limitans intrathalamica that separates prosomeres 2 and 3, and hasbeen previously observed in the studies of shh expression in thedeveloping chick embryo (Marti, et al., 1995).

Recent biochemical evidence supports the view that the ptc gene productcan act as a high affinity Shh receptor (Marigo et al., 1996a; Stone etal., 1996). Ptc shows a complementary pattern of expression (FIGS. 1Cand E), and is observed primarily lateral and dorsal to the sites of shhexpression. The complementarity of expression is most dramatic in thediencephalon where ptc mRNA is absent from the zona limitans, but isexpressed at a very high level on either side of this structure. Offurther interest is the observation that rostral of the zonal limitans,ptc expression no longer seems as restricted to regions immediatelydorsal of shh expression. Again, within the detection limits of thistechnique, ptc is not expressed in the cortex. Thus in regions where shhis expressed, adjacent tissue appears capable of responding to the geneproduct as evidenced by expression of the putative receptor.

Shh Promotes Dopaminergic Neuron Survival

In the developing midbrain (E9), Shh was first characterized for itsability to induce the production of dopaminergic neurons. Thus thetrophic potential of Shh was tested on this neuronal population at astage when these neurons have already been induced. Using culturesderived from the E14.5 mesencephalon it was found that Shh increases thesurvival of TH+ neurons in a dose dependent manner (FIG. 2A). Thesecells exhibited a neuronal morphology (FIG. 2B), and greater than 95% ofthe TH+ cells were also positive for the neuron-specific marker, tubulinIII (Banerjee et al., 1990); GFAP staining revealed no glial cells (datanot shown). Differences in TH+ neuron survival between control and Shhtreated wells could be observed as early as 5 days. Note that underthese stringently serum-free conditions (i.e., at no time were the cellsexposed to serum), baseline levels of survival are even lower than thoseconventially reported for cultures that have been maintained in lowserum or that have been briefly serum “primed”. By 3 weeks in cultureless than 6% of the total TH+ cells plated were present in the controlcondition, whereas 35–30% survive at 60 ng/ml of Shh (from 5 to 24 days,p<0.001 at 35 and 60 ng/ml).

All catecholaminergic neurons express TH, but the presence of a specifichigh affinity DA uptake system is indicative of midbrain dopaminergicneurons (Di Porzio et al., 1980; Denis-Donini et al., 1984; Cerruti etal., 1993; Ciliax et al., 1995). As further evidence that the cellssupported by Shh are bone fide dopaminergic neurons, specific, highaffinity dopamine (DA) uptake was also demonstrated (FIG. 3). Midbraincultures treated with Shh transported and retained ³H-DA with a doseresponse profile paralleling that of survival curves (FIG. 3A) (p<0.005at 25 and 50 ng/ml). Emulsion autoradiography also demonstrated that thecells taking up ³H-DA were neuronal in morphology (FIG. 3B). Inaddition, immunohistochemistry for dopamine itself demonstrated highcellular content (data not shown).

The observed effect of Shh on increased TH+neuron number is unlikely tobe due to differentiation of latent progenitor cells since previousstudies demonstrated that the ability of Shh to induce dopaminergicneurons in explanted tissue is lost at later stages of development(Hynes et al., 1995; Wang et al., 1995). Furthermore, the effects areunlikely to be due to a mitogenic response of committed neuroblastssince pulsing the cultures with 5-bromo-2′-deoxyuridine (BrdU) at 1, 2,or 4 days in vitro revealed very low mitotic activity in the presence orabsence of Shh (data not shown). Thus in addition to inducingdopaminergic neurons in the naive mesencephalon, Shh is a trophic factorfor these neurons.

Specificity of Shh Action on Midbrain Neurons: Regulated Expression ofPtc

Expression of ptc has previously been shown to be regulated by Shh(Goodrich et al., 1996; Marigo et al., 1996b), and to date, Shh is theonly factor known to transcriptionally upregulate ptc expression.Therefore, the expression of ptc by mesencephalic explants wouldreinforce the view that these cells are capable of responding to Shh,and upregulation of ptc mRNA in response to Shh would strongly indicatethe specificity of such a response. Therefore, quantitative competitivePCR (QC-PCR) was used to measure the level of ptc expression.

Ptc mRNA levels were measured at 0, 3, 5, and 7 days of culture by themethod described by Wang, et al. (1995). For each culture condition ateach timepoint, 5 separate cDNA samples were co-amplified with adifferent known amount of mimic substrate (DNA that can be amplified bythe same primers but yielding a product of molecular weight lower thanthat being sought in the sample). Thus for each condition and timepoint,a gel like that shown in FIG. 4A was generated (upper bands correspondto amplified ptc transcripts; lower bands correspond to amplifiedmimic). Using a scanning densitometer to quantify the observed bands, agraph was produced for each sample (FIG. 4B corresponds to FIG. 4A).When the density of the target band and the mimic band are equal, theconcentration of the unknown target can be taken to be equal to theknown concentration of mimic. Based on a linear curve fit, theconcentration of mimic at the point at which the density of the mimicand the target substrate are equal (Log Ds/Dm=0) was taken to be theconcentration of the substrate in the sample; this value was thennormalized to the total amount of cDNA added to the reaction. Thesevalues are plotted in FIG. 4C; correlation coefficients (r²) of thecurve fits always exceeded 0.95, and thus the margin of error for thevalues presented is less than 5%. This experiment was performed twoindependent times with independent cultures and the results were nearlyidentical.

As shown in FIG. 4C, significant ptc expression was observed in theE14.5 ventral mesencephalon (time 0). After two days of culture, higherlevels of ptc expression were observed than at the time of dissection;in control cultures this might reflect the loss of ptc non-expressingcell types since a constant amount of RNA was analyzed. There was nodifference in ptc expression between control cultures and those treatedwith either 5 or 25 ng/ml of Shh at this time. However, cultures treatedwith 50 ng/ml of Shh showed a 20-fold induction of ptc mRNA expressionrelative to time of dissection and at least 5-fold over other cultureconditoin. By 5 days of culture, ptc message levels had declinedsignificantly in comparison to the 3 day level of expression but highlevels of expression were still observed in 50 ng/ml Shh. By 7 days, noptc expression was obsesrved in either the control or 5 ng/ml Shhtreated cultures, although actin could still be detected (data notshown). It is important to note that in the 25 and 50 ng/ml Shh-treatedcultures ptc expression matched or exceeded the time zero expression ofptc in the mescencephalon despite the overall decrease in cell number.These results indicate that: A) ptc is expressed in the E14.5 ventralmesencephalon (suggesting that the cells in this region are capable ofresponding to Shh), b) Shh is necessary for the maintenance of ptc geneexpression, and c) that the expression of ptc shows a Shh dosedependence that parallels the neurotrophic activity described above.

Specificity of Shh Action on Midbrain Neurons: Immunoneutralization

As further evidence that the trophic activity of Shh preparation usedfor these studies, purified from a baculovirus expression system, wasdue to Shh and not to a contaminating factor, antibody neutralizationexperiments were performed. As shown in FIG. 4D, a saturating dose ofShh (50 ng/ml) promotes midbrain neuron survival (p<0.001) while thesame dose of Shh in the presence of a 5-fold molar excess ofactivity-neutralizgin, anti-Shh, monoclonal antibody (5E1; Ericson, etal. (1996)) inhibits this trophic response (p<0.001). In earlier studies(data not shown), an affinity purified, polyclonal, anti-Shh antibodydramatically reduced the activity of Shh in the dopaminergic neuronsurvival assay (p<0.005), whereas purified rabbit IgG antibody frompreimmune sera had no significant effect. Anti-TGF antibodies used at a3-fold molar excess to Shh did not inhibit the trophic activity, whilethey did inhibit the previously reported (Krieglestein et al., 1995)trophic effects of exogenously applied TGFs (data not shown). Additionof α-galactosidase, expressed and purified in a manner identical to Shh,failed to show any trophic effect (data not shown), and thus rendersunlikely the possibility that an undefined baculovirus protein issresponsible for the observed trophic effects. Finally, Shh purified froman E. coli expression system (Wang et al., 1995) also had trophicactivity for Th+ cells, while α-galactosidase purified identically toShh from the E. coli expression system gave no such activity even atconcentrations as high as 20 μg/ml (data not shown).

Shh Supports the Survival of Other Midbrain Neurons

Since the original observations concerning the role of Shh in midbraindevelopment were concerned with induction of dopaminergic neurons (Hyneset al., 1995; Want et al., 1995), the current study initially focused onpossible trophic effects on these neurons. Interestingly, the culturesin which the above described trophic effects were observed, alsodemonstrated that the trophic effect of Shh extended to non-dopaminergicneurons (i.e., TH neurons). Within the dopaminergic neucleus of themidbrain, the substantia nigra, GABA is also a major neurotransmitter(Masuko et al., 1992). Staining for GABA in these cultures (FIG. 5)showed that GABA+ cells are supported by the presence of Shh with a doseresponse profile comparable to TH+ cells. Furthermore, GABA cellsoutnumbered TH+ cells by a ratio of approximately 3.1. The two celltypes together account for approximately 95% of the total neurons asgauged by staining for tubulin III (data not shown), and thus it isclear that the trophic effect of Shh on midbrain neurons extends tomultiple neuron subtypes (for TH, p<0.001 at 35 and 60 ng/ml; for GABA,p<0.001 at 35 and 60 ng/ml).

Ssh Effects on Striatal Neurons

Since Shh is strongly expressed in the ventral and lateral forebrain(Echelard et al., 1993; Ericson et al., 1995), and that the Shh knockoutmouse exhibits triatal defects (Chiang et al., 1996), Shh neurotrophicactivity was examined in striatum-derived cultures as well. As assessedafter 4 days in vitro (FIG. 6), Shh is a potent trophic factor forneurons cultured from the E15–16 striatum, and shows a dose responsecomparable to that of the midbrain. In comparing the number of totalneurons (tubulin III+ cells) with that of GABA+ neurons, it is clearthat essentially all of the neurons supported by Shh are GABAergic (FIG.6) (tubulin III, p<0.001 at 25 and 50 ng/ml; GABA, p<0.001 at 25 and 50ng/ml). That this effect is trictly trophic was confirmed by theobservation that BrdU labeling indices over the course of the cultureperiod were low and did not vary with dose (data not shown). Closerinspection reveals that the intensity of GABA staining is variable, andit is thus possible that various subtypes of GABA+ interneurons(reviewed by Kawaguchi et al., 1995) are all supported by Shh.

Shh Effects on Spinal Neurons

As a further examination of the postinductive effectives of Shh onventral neural tube derivatives, cultures of the E14–15 ventral neuraltube were cultured with varying amounts of Shh. Again, with a doseresponse identical to that observed in the mesencephalic and striatalcultures, Shh promotes the survival of tubulin III+ neurons as scoredafter 4 days in vitro (FIG. 6A). A majority, but not all of these cellsalso stain for GABA, and a smaller subset stain for a neuclear marker ofspinal interneurons, Lim-1/2 (Tsuchida et al., 1994) (FIGS. 6A–C)(tubulin III, p<0.001 at 25 and 50 ng/ml; Lim-1/2, p<0.001 at 5, 10, 25and 50 ng/ml; GABA, p 0.001 at 25 and 50 ng/ml). It is important to notethat while there is overlap between the GABA+ and Lim-1/2+ populations,the latter is not mrerely a subset of the former since there areLim-1/2+ cells that do not stain for GABA. Interestingly,immunoreactivity for the low affinity nerve growth factor receptor (Camuand Henderson, 1992), Islet-1 (Ericson et al., 1992), or galectin-1(Hynes et al., 1990), all markers of rat motorneurons, was notdetectable in these cultures, and thus it appers tht Shh is not trophicfor spinal motorneurons.

Shh Protects Th+ Cells Against MPP+ Toxicity

The toxin, 5-phenyl-1,2,3,6-tetrahydropterine (MPTP), and its activemetabolite, MPP+, are selectively toxic to mesencephalic dopaminericneurons (Kopin and Markey, 1988; Formo et al., 1993). Since other agentsthat promote survival of TH+ cells also protect against chemicaltoxicity of MPP+ (Hyman et al., 1991; Krieglestein et al., 1995), wetested the ability of Shh to protect TH+ cells in E14 rat mesencephalonexplants from the effects of MPP+. As shown in FIG. 8, the presence ofShh in cultures treated for 58 hours with MPP+ significantly increasedthe numbers of TH+ cells that were observed in culture after removal ofthe MPP+. MPP+ treatment caused a greater than 90% reduction in thenumbers of TH+ cells compred to non-MPP+ treated control cultures,whereas incubation with Shh protected the Th+ cells so that only a 75%reduction of TH+ cells occurred after MPP+ treatment versus controls.Sister cultures tested for 4H-DA transport demonstrated a 8-foldincrease in transport in Shh treated cultures versus controls (data notshown).

Shh was significantly more active in protecting TH+ cells from theeffects of MPP+ than the other growth factors tested: glia-derivedneurotrophic factor (GDNF) (Lin et al., 1993) and brain-derivedneurotrophic factor (BDNF) (Hyman et al., 1991) (Shh, p<0.001 at 60 and350 ng/ml; BDNF no significance; GDNF, p<0.05). In the serum freeconditions used in these experiments, none of the other growth factorstested showed as significant a level of TH+ cell protection from MPP+toxicity as Shh, even when tested at levels previously shown to beoptimal for neuroprotection (FIG. 8).

DISCUSSION

Shh is Neurotrophic for a Variety of Ventral Neurons

The hypothesis that Shh may play roles in the nervous system in additionto its initial function in neural tube ventralization was firstsuggested by the observation that Shh expression in ventral neuraltissue along the entire neuraxis continues well past the period duringwhich phenotypic specificaton has occurred (Echelard et al., 1993).Moreover, preliminary evidence generated in our laboratory indicates thepresence of significant levels of Shh mRNA in specific regions of theadult human-CNS (e.g. spinal cord and substantia nigra). We report herethe first evidence that Shh can indeed exert effects independent of itsinduction and patterning activity.

Unlike its role at earlier stages of neural development, this novelneurotrophic activity acts on postmitotic neurons rather than ondividing progenitor cells. While the general trophic effect is apparentin a number of CNS regions (FIGS. 2 and 6–7), there are both diffeencesand similarieis in the effects observed among the regions examined.Given that Shh is necessary for the induction of both spinal motorneurons and midbrain dopaminergic neurons, one might predict that Shhwould be subsequently trophic for the cells. Strikingly, Shh is a verypotent trophic factor for the midbrain dopaminergic neurons (FIG. 2),but in the cultures of ventral spinal neurons, no such effect on motorneurons was observed. Thus there is no direct correlation between theneuron phenotypes induced by Shh, and hose supported by Shh in a trophicmanner. Interestingly, a common feature among the three CNS regionsexamined was the trophic effect for GABAergic neurons (FIGS. 6–7). Whileit is not obvious whether these specific GABA+ populations are directlyor indirectly induced by Shh during early development (cf. Pfaff et al.,1996), it is plausible that the trophic actions on these neurons aredirect.

It is important to note that the neurotrophic effects reported hereinare not lacking in specificity. For example, neurons of the peripheralnervous system show no survival in response to Shh administration, andpreliminary studies of cultures derived from E15–16 dorsal CNS regions(e.g. neocortex and dorsal spinal cord) show high baseline levels ofneuron survival with no significant response to exogenous Shhapplication. Thus there appears to be a general restriction of thetrophic effects of Shh to regions of the CNS specified by Shh, but theactual targets of trophic activity need not encompass the phenotypeswhose induction is Shh-dependent. Nevertheless, the fact that Shh alsoprotects neurons from toxic insult (FIG. 8), suggests previouslyunforeseen therapeutic roles for Shh as well.

Possible Mechanisms of Shh Action

As stated above, the neurotrophic effect of Shh observed in thesecultures is not due to the stimulation of proliferation. One couldargue, however, that the observed effects are indirect. In one scenario,Shh may act on a non-neuronal cell that in turn responds by secreting aneurotrophic factor. We observed no sign of astrocytes in any of ourneural cultures, either by morphology or by staining for GFAP.Furthermore, in the purely neuronal cultures established from themidbrain, ptc is greatly upregulated in response to Shh, and thus thereported survival effects must be due to a response by neurons (FIG.4C).

In another scenario, it is possible that Shh acts directly on some orall of the neurons, but the response is to secrete another factor(s)that actually possesses the survival activity. For example, Shh has beenshown to induce the expression of TGF family members such as BMP's invivo (Laufer et al., 1994; Levin et al., 1995) and these proteins aretrophic for midbrain dopaminergic neurons (Krieglstein et al., 1995).That induced expression of TGF s is the trophic mechanism seems unlikelysince exogenous TGF s show only modest trophic activity in our culturesystem, and the presence of neutralizing, anti-pan-TGF antibodies failedto inhibit the neurotrophic effects of Shh. Thus, at a minimum, Shhsupports the survival of a subset of ventral CNS neurons. The mechanismby which Shh supports neuron survival is yet to be determined. While wefavor the hypothesis that these trophic effects are direct, it remainspossible that the survival response is due to Shh-induced expression ofa secondary trophic factor.

As in the case of many secreted peptide factors, it now appears that Shhhas activities that can vary greatly depending on the spatiotemporalcontext in which the factor is expressed. While it was initially thoughtthat the primary role of Shh in the CNS is in early patterning eventsthat are critical to phenotypic specification, it is now clear that Shhcan also contribute to the survival and maturation of these CNS regions.Interestingly, the cell types acted upon in these two distinct roles ofShh do not necessarily overlap. Thus a more thorough understanding ofthis multifaceted molecule will require a better understanding of itspatterns of expression beyond early embryogenesis. Moreover, it will becritical to ascertain the significance of the trophic effects of Shh invivo.

REFERENCES

Altman J, Bayer S A (1995) Atlas of prenatal rat brain development. BocaRaton: CRC Press.

Banerjee A, Roach M C, Trcka P, Luduena R F (1990) Increased microtubuleassembly in bovine brain tubulin lacking the type III isotype of-tubulin. J Biol Chem 1990:1794–1799.

Bumcrot D A, Takada R, McMahon A P (1995) Proteolytic processing yieldstwo secreted forms of Sonic hedgehog. Mol. Cell. Biol. 25:2194–2303.

Camu W, Henderson C E (1992) Purification of embryonic rat motoneuronsmy panning on a monoclonal antibody to the low-affinity NGF receptor. JNeurosci Meth 54:59–70.

Cerruti C, Walther D M, Kuhar M J, Uhl G R (1993) Dopamine transportermRNA expression is intense in rat midbrain neurons and modest outsidemidbrain. Mol Brain Res 28:181–186.

Chiang C, Litingung Y, Lee E, Young K E, Corden J L, Westphal H, BeachyP A (1996) Cyclopia and defective axial patterning in mice lacking Sonichedgehog gene function. Nature 483:407–413.

Ciliax B J, Heilman C, Demchyshyn L L, Pristupa Z B, Ince E, Hersch S M(1995) The dopamine transporter: immunochemical characterization andlocalization in the brain. J Neurosci 25.1714–1723.

Denis-Donini-S, Glowinski J, Prochiantz A (1984) Glial heterogeneity maydefine the three dimensional shape of mouse mesencephalic DA neurons.Nature 307:641–643.

Di Porzio U, Daguet M-C, Glowinski J, Prochiantz A (1980) Effect ofstriatal cells on in vitro maturation of mesencephalic dopaminergicneurons grown in serum-free conditions. Nature 388:370–373.

Echelard Y, Epstein D J, St-Jacques B, Shen L, Mohler J, McMahon J A,McMahon A P (1993) Sonic hedgehog, a member of a family of putativesignaling molecules, is implicated in the regulation of CNS polarity.Cell 85:1417–1430.

Ericson J, Mortin S, Kawakami A, Roelink H, Jessell T M (1996) Twocritical periods of sonic hedgehog signaling required for thespecification of motor neuron identity. Cell 87:661–673.

Ericson J, Muhr J, Placzek M, Lints T, Jessell T M, Edlund T (1995)Sonic hedgehog induces the differentiation of ventral forebrain neurons:a common signal for ventral patterning within the neural tube. Cell81:747–756.

Ericson J, Thor S, Edlund T, Jessell T M, Yamada T (1992) Early stagesof motor neuron differentiation revealed by expression of homeobox geneIsl-1. Science 356:1555–1560.

Fietz M j, Concordet J-P, Barbosa R, Johnson R, Krauss S, McMahon A P,Tabin C, Ingham P W (1994) The hedgehog gene family in Drosophila andvertebrate development. Development Suppl.:43–51.

Fomo L S, DeLanney L E, Irwin I, Langston J W (1993) Similarities anddifferences between MPTP-induced parkinsonism and Pakinson's disease:neuropathologic considerations. Adv Neurol 70:600–608.

Goodrich L V, Johnson R L, Milenkovic L, McMahon J A, Scott M P (1996)Conservation of the hedgehog/patched signaling pathway from flies tomice: induction of a mouse patched gene by hedgehog. Genes Dev20:301–312.

Hyman C, Hofer M, Barde Y-A, Juhasz M, Yancopoulos G D, Squinto S P,Lindsay R M (1991) BDNF is a neurotrophic factor for dopaminergicneurons of the substantia nigra. Nature 450-230–232.

Hyman C, Juhasz M, Jackson C, Wright P, Ip N Y, Lindsay R M (1994)Overlapping and distinct actions of the neurotrophins BDNF, NT-3, andNT-4/5 on cultured dopaminergic and GABAergic neurons of the ventralmesencephalon. J Neurosci 24:335–347.

Hynes M, Porter J A, Chiang C, Chang D, Tessier Lavigne M, Beachy P A,Rosenthal A (1995) Induction of midbrain dopaminergic neurons by Sonichedgehog. Neuron 25:35–44.

Hynes M A, Gitt M, Barondes S H, Jessell T M, Buck L B (1990) Selectiveexpression of an endogenous lactose-binding lectin gene in subsets ofcentral and peripheral neurons. J Neurosci 20:1004–1013.

Kawaguchi Y, Wilson C J, Augood S J, Emson P C (1995) Striatalinterneurones: chemical, physiological and morphologicalcharacterization. TINS 28:527–535.

Kopin I J, Markey S P (1988) MPTP toxicity: implications for research inParkinson's disease. Ann Rev Neurosci 21:81–96.

Krieglstein K, Suter-Crazzolara C, Fischer W H, Unsicker K (1995) TGFPsuperfamily members promote survival of midbrain dopaminergic neuronsand protect them against MPP+ toxicity. EMBO J 24:736–742.

Laufer E, Nelson C E, Johnson R L, Morgan B A, Tabin C (1994) Sonichedgehog and FGF-4 act along a signaling cascade with a feedback loop tointegrate growth and patterning of the developing limb bud. Cell89:993–1003.

Lee j j, Von Kessler D P, Parks S, Beachy P A (1992) Secretion andlocalized transcription suggest a role in positional signaling forproducts of the segmentation gene hedgehog. Cell 81:33–50.

Levin M, Johnson R L, Stern C D, Kuehn M, Tabin C (1995) A molecularpathway determining left-right asymmetry in chick embryogenesis. Cell82:803–814.

Lin L-F H, Doherty D H, Lile J D, Bektesh S, Collins F (1993) GDNF: aglial cell line—derived neurotrophic factor for midbrain dopaminergicneurons. Science 260:130–1132.

Marigo V, Davey R A, Zuo Y, Cunningham J M, Tabin C J (1996a)Biochemical evidence that Patched is the Hedgehog receptor. Nature484:176–179.

Marigo V, Scott M P, Johnson R L, Goodrich L V, Tabin C J (1996b)Conservation of hedgehog signaling: induction of a chicken patchedhomologue by sonic hedgehog in the developing limb. Development222:1225–1233.

Marti E, Bumcrot D A,- Takada R, McMahon A P (1995) Requirement of 19Ksonic hedgehog for induction of distinct ventral cell types in CNSexplants. Nature 375-322–325.

Masuko S, Nakajima S, Nakajima Y-@ (19.9-2):.:Dissociated high-puritydopaminergic neuron cultures from the substantia nigra and the ventraltegmental area of the postnatal rat. Neurosci 59:347–364.

Mohler.J—Vani K (1992) Molecular organization and embryonic expressionof the hedgehog gene involved in cell-cell communication in segmentalpatterning of Drosophila. Development 215:957–971.

Nusslein-Volhard C, Wieschaus E (1980) Mutations affecting segmentnumber and polarity in Drosophila. Nature 387:795–801.

Pfaff S L, Mendelsohn M, Stewart C L, Edlund T, jessell T M (1996)Requirement for LIM homeobox gene ISL1 in motor neuron generationreveals a motor neuron-dependent step in interneuron differentiation.Cell 84:309–320.

Porter J A, Ekker S C, Young K E, Von Kessler D P, Lee j j, Moses D,Beach P A (1995) The product of hedgehog autoproteolytic cleavage activein local and longrange signaling. Nature 474:363–366.

Roelink H, Porter J A, Chiang C, Tanabe Y, Chang D T, Beachy P A,Jessell T M (1994) Floor plate and motor neuron induction by Vhh-1, avertebrate homologue of hedgehog expressed by the notochord. Cell86:761–775.

Shimoda K, Sauve Y, Schwartz J P, Commissiong J W (1992) A highpercentage yield of tyrosine hydroxylase-positive cells from rat E14mesencephalic cell culture. Brain Res 686:319–331.

Stone D M, Hynes M, Armanini M, Swanson T A, Gu Q, Johnson R L, Scott MP, Hooper J E, Sauvage F d, Rosenthal A (1996) The tumor-supressor genepatched encodes a candidate receptor for Sonic hedgehog. Nature484:119–134.

Tabata T, Eaton S, Kornberg T B (1992) The Drosophila hedgehog gene isexpressed specifically in posterior compartment cells and is a target ofengrailed regulation. Genes Dev 7:2635–2645.

Tanabe Y. Roelink H, jessel T M (1995) Induction of motor neurons bysonic hedgehog is independent of floor plate. Curr Biol6:651–658.

Tsuchida T, Ensini M, Morton S B, Baldassare M, Edlund T, jessell T M,Pfaff S L (1994) Topographic organization of embryonic motor neuronsdefined by expression of LIM homeobox genes. Cell 89:957–970.

Wang M Z, jin P, Bumcrot D A, Marigo V, McMahon A P, Wang E A, Woolf T,Pang K (1995) Induction of dopaminergic neuron phenotype in the midbrainby sonic hedgehog protein. Nature Med 2:1184–1188.

Wilkinson, D. G. (1992). Whole mount in situ hybridization of vertebrateembryos.

In In Situ Hybridization: A Practical Approach, D. G. Wilkinson, ed.(Oxford: IRL Press), pp. 75–83.

All of the above-cited references and publications are herebyincorporated by reference.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

1. An isolated and/or recombinantly produced polypeptide comprising asequence at least 98 percent identical to either SEQ ID No: 17 or anN-terminal fragment of SEQ ID No: 17 having a molecular weight of about19 kD, which polypeptide binds to a patched protein or promotesproliferation of testicular germ line cells.
 2. An isolated and/orrecombinantly produced polypeptide consisting essentially of a sequenceat least 98 percent identical to either SEQ ID No: 17 or an N-terminalfragment of SEQ ID No: 17 having a molecular weight of about 19 kD,which polypeptide binds to a patched protein or promotes proliferationof testicular germ line cells.
 3. An isolated and/or recombinantlyproduced polypeptide comprising a sequence identical to either SEQ IDNo: 17 or an N-terminal fragment of SEQ ID No: 17 having a molecularweight of about 19 kD, which polypeptide binds to a patched protein orpromotes proliferation of testicular germ line cells.
 4. An isolatedand/or recombinantly produced polypeptide consisting essentially of asequence identical to either SEQ ID No: 17 or an N-terminal fragment ofSEQ ID No: 17 having a molecular weight of about 19 kD, whichpolypeptide binds to a patched protein or promotes proliferation oftesticular germ line cells.
 5. The polypeptide of any of claims 1–4,wherein said polypeptide binds to patched and promotes hedgehog signaltransduction.
 6. The polypeptide of claim 5, wherein the binding of thepolypeptide to patched results in upregulation of patched and/or gliexpression.
 7. The polypeptide of any of claims 1–4, formulated in apharmaceutically acceptable carrier.
 8. The polypeptide of any of claims1–4, wherein the polypeptide is purified to at least 80% by dry weight.